JP2017525388A - How to produce acetoin - Google Patents

Abstract

  The present invention relates to a recombinant yeast having reduced pyruvate decarboxylase activity, wherein the following are inserted in the genome: one or more nucleic acids encoding acetolactate synthase or ALS, One or more nucleic acids encoding lactate decarboxylase or ALD, and one or more copies of nucleic acid encoding NADH oxidase or NOXE.

Description

  The present invention relates to microorganisms having an improved acetoin pathway. Recombinant microorganisms are modified to improve acetoin production compared to unmodified microorganisms. The present invention also provides a method for producing acetoin using such a microorganism.

  Acetoin, also known as 3-hydroxybutanone or acetylmethylcarbinol, is formed in bacteria by the action of two enzymes from pyruvate, that is, the two pyruvate molecules An α-acetolactic acid synthase that catalyzes a condensation in a single decarboxylation to give α-acetolactic acid and an α-acetolactic acid decarboxylase that decarboxylates this last to acetoin (Juni, 1952). ).

  Acetoin is a flavoring agent commonly used in the food industry and has replaced diacetyl because it is considered safer (Huang, 2011). Acetoin is used as a flavoring ingredient in formulations for raspberry, strawberry, vanilla, walnut, lamb, butter, butterscotch, caramel, coconut, coffee and fruit flavors. Acetoin may be added to alcoholic and non-alcoholic beverages such as cream soda. The buttery flavor is well suited for ice cream, ice cream, candy, baked goods, margarine, gelatin dessert, cottage cheese, margarine and shortening. Acetoin is used in fragrances in the beauty industry and in fragrances as an aroma carrier. Finally, acetoin is one of the top chemical additives as a flavoring agent in tobacco and in electronic tobacco. In addition, acetoin is a precursor of methyl vinyl ketone (MVK), a useful intermediate in chemistry.

  The traditional chemical synthesis of acetoin faces the challenges of oil shortages and environmental pollution, while the market for acetoin as a food flavorant currently grows at an annual rate of 5 to 5,5% And is expected to reach $ 14 billion in 2018. The fragrance market is expected to exceed $ 16 billion in 2018.

  Many chemicals that could only be produced by traditional chemical processes in the past can now have the potential to be biologically generated using renewable resources (Danner & Braun, 1999; Hatti- Kaul et al., 2007). The microbial production of acetoin is one such example. Interest in this bioprocess has increased significantly because acetoin has numerous industrial applications, as described above, and microbial production has become an oil supply for the production of platform chemicals. This is because it is expected to ease the dependency of Saccharomyces cerevisiae is a particularly well-suited platform for such bioprocesses (Nielsen 2013). With respect to microbial production of acetoin, most studies used microorganisms such as Candida glabrata, Bacillus subtilis to produce acetoin (Shubo Li et al., Microbial Cell Factories 2014, 13:55; Silbersack J et al., Appl Microbiol Biotechnol. 2006 Dec; 73 (4): 895-903;

  Therefore, acetoin production by GRAS (ie, generally recognized as safe) microorganisms would be desirable. Yeast, and more particularly budding yeast, are suitable microorganisms in this regard. Saccharomyces cerevisiae is known to naturally produce acetoin, but the yield and productivity of acetoin production are poor. Ethanol production is actually the most obvious barrier to efficient acetoin production in Saccharomyces cerevisiae because the key intermediate pyruvic acid is preferentially used to produce ethanol rather than acetoin Because it is done.

International Publication No. 2004/076659 US Patent No. 2011/0124060 International Publication No. 2013/076144 International Publication No. 2006/134277 International Publication No. 2011/041426

Shubo Li et al., Microbial Cell Factories 2014, 13:55 Silbersack J et al., Appl Microbiol Biotechnol. 2006 Dec; 73 (4): 895-903 DiCarlo et al. (Nucl. Acids Res., Vol. 41, No. 7, 2013: 4336-4343) D. Burke et al., Methods in yeast Genetics-A cold spring harbor laboratory course Manual (2000) Casal M, et al. (1999), J. Bacteriol., 181 (8): 2620-3 Tanaka et al. (2012) Appl Environ Microbiol., 78 (22): 8161-3 Shao et al. (Nucleic Acids Research, 2009, Vol. 37, No. 2: e16) Shao et al. (Methods in Enzymology, 2012 Elsevier Inc., Vol. 517: 203 Wang et al. (Biochemistry, 2001, 40: 1755-1763) Poulsen et al. (Eur. J. Biochem. 185, 1989: 433-439) Dulieu et al. (Enzyme and Microbial Technology 25, 1999: 537-542) Lopez DE FELIPE et al. (International Daily Journal, 2001, vol. 11: 37-44 (ISSN 0958-6946)) velculescu et al. (1997) Cell 88, 243-251. Blazeck & Alper (2013) Biotechnol. J. 8 46-58 Yamanishi et al. (2013) ACS synthetic biology 2, 337-347 buscke et al., biosource technology 2013 Kim et al. (Bioresource Technology, vol. 146, 2013: 274) Kim et al. (Journal of Biotechnology, 2014) Thomas and Rothstein (1989), Cell. 56, 619-630 Methods in yeast Genetics-A cold spring harbor laboratory course Manual (2000) by D. Burke, D. Dawson, T. Stearns CSHL Press Gonzales et al. (2010), Applied and environmental Microbiology 76 670-679

  Thus, for obvious reasons, improving acetoin production through microbial processes, and more particularly, converting pyruvate to acetoin remains an ongoing challenge. More particularly, there is still a need for stable recombinant microorganisms with enhanced production yields of acetoin that are particularly compatible with industrialization requirements.

The present invention relates to a recombinant yeast having reduced pyruvate decarboxylase activity in its genome:
-One or more nucleic acids encoding acetolactate synthase or ALS,
-One or more nucleic acids encoding acetolactate decarboxylase or ALD, and
-One or more copies of nucleic acid encoding NADH oxidase (NOXE) has been inserted.

According to a particular embodiment, the recombinant yeast according to the invention has the following formula:
(I) 5 '-[gene 1] _x1 -3' and 5 '-[gene 2] _x2 -3' and 5 '-[gene 3] _x3 -3',
(II) 5 '-[gene 1] _x1- [gene 2] _x2 -3' and 5 '-[gene 3] _x3 -3',
(III) 5 '-[gene 1] _x1- [gene 2] _x2- [gene 3] _x3 -3', and combinations thereof,
(Where:
-"Gene 1" means a nucleic acid selected from the group comprising ALS, ALD or NOXE;
-"Gene 2" means a nucleic acid selected from the group comprising ALS, ALD or NOXE, but different from gene 1;
-"Gene 3" means a nucleic acid selected from the group comprising ALS, ALD or NOXE, but different from genes 1 and 2;
-"ALS" is a nucleic acid encoding acetolactate synthase;
-"ALD" is a nucleic acid encoding acetolactate decarboxylase;
-"NOXE" is a nucleic acid encoding NADH oxidase;
-Each of "x1", "x2" and "x3", independently of one another, represents an integer in the range of 0 to 50, preferably 0 to 20,
However, the recombinant yeast contains at least one nucleic acid encoding each of ALS, ALD and NOXE)
One or more DNA constructs selected from the group comprising:

  Preferably, each of “x1”, “x2” and “x3”, independently of each other, is 0 to 12, more particularly in the range of 0 to 5, in particular including the range of 0 to 3, 0 to 15. Represents an integer in the range, and better still represents an integer equal to 1.

  According to another particular embodiment, the recombinant yeast according to the invention is at least one, preferably at least two, DNA constructs of formula (II) as defined above, wherein “gene 3” is NADH oxidase. A DNA construct may be included which means a nucleic acid encoding.

According to yet another specific embodiment, the recombinant yeast according to the invention is at least one, preferably at least two DNA constructs of formula (IIa), which are the same or different, wherein each formula (IIa) is formula:
(IIa) 5 '- [( prom5) y1 - Gene 1-term5] _x5 - [prom1- gene 1-term1] _x1 - [prom2- gene _{2- (term2) z1] x2 -3} ' and 5 '- [( prom3) _y2 -gene 3- (term3) _z2 ] _x3 -3 '
(Where:
-Gene 1, gene 2, gene 3, `` x1 '', `` x2 '' and `` x3 '' are, for example, those defined above;
-"X5" represents an integer equal to 0 or 1;
-`` Y1 '', `` y2 '', `` z1 '' and `` z2 '' represent, independently of one another, an integer equal to 0 or 1;
-When the recombinant yeast comprises at least two DNA constructs of formula (IIa), `` x1 '', `` x2 '', `` x3 '', `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '''' May be the same or different;
-"Prom 1" is a regulatory sequence that controls the expression of the sequence encoding gene 1;
-"Prom 2" is a regulatory sequence that controls the expression of the sequence encoding gene 2;
-"Prom 3" is a regulatory sequence that controls the expression of the sequence encoding gene 3;
-`` Prom 5 '' is a control sequence that controls the expression of gene 1, said prom 5 being the same as or different from prom 1;
-"Term 1" is a transcription terminator sequence that terminates the expression of the sequence encoding gene 1;
-"Term 2" is a transcription terminator sequence that terminates the expression of the sequence encoding gene 2;
-"Term 3" is a transcription terminator sequence that terminates the expression of the sequence encoding gene 3;
-"Term 5" is a transcription terminator sequence that terminates the expression of gene 1, and said term 5 is the same as or different from term 1)
A DNA construct having

According to another particular embodiment, the recombinant yeast according to the invention is the same or different, at least one, preferably at least two DNA constructs of formula (IIb), each formula (IIb) having the formula :
(IIb) 5 '- [( prom5) y1 -ALS-term5] x5 - [prom1-ALS-term1] x1 - [prom2-ALD- (term2) z1] x2 -3' and 5 '- [(prom3) _y2 -NOXE- (term3) _z2 ] _x3 -3 '
(Where:
-ALS, ALD, NOXE; `` x1 '', `` x2 '', `` x3 '', `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '' are, for example, those defined above;
-If said recombinant yeast comprises at least two DNA constructs of formula (IIb), `` x1 '', `` x2 '', `` x3 '', `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '' May be the same or different;
-"Prom 1" is a regulatory sequence that controls the expression of the sequence encoding acetolactate synthase;
-"Prom 2" is a regulatory sequence that controls the expression of the sequence encoding acetolactate decarboxylase;
-"Prom 3" is a regulatory sequence that controls the expression of the sequence encoding NADH oxidase;
-"Prom 5" is a control sequence that controls the expression of a sequence encoding acetolactate synthase, said prom 5 being the same as or different from prom 1;
-"Term 1" is a transcription terminator sequence that terminates the expression of the sequence encoding acetolactate synthase;
-"Term 2" is a transcription terminator sequence that terminates the expression of the sequence encoding acetolactate decarboxylase;
-"Term 3" is a transcription terminator sequence that terminates the expression of the sequence encoding NADH oxidase;
-"Term 5" is a transcription terminator sequence that terminates the expression of the sequence encoding acetolactate synthase, and said term 5 is the same as or different from term 1)
A DNA construct having

  According to another particular embodiment, the recombinant yeast according to the invention may comprise at least two DNA constructs of the formula (II), (IIa) or (IIb) provided that all of the NOXE nucleic acids are present. The copy is located in one of at least two DNA constructs of formula (II), (IIa) or (IIb).

According to another particular embodiment, the recombinant yeast according to the invention has at least two, preferably exactly two, the following formulas (IIc) and (IId):
(IIc) 5 '- [( prom5) y1 -ALS-term5] x5 - [prom1-ALS-term1] x1 - [prom2-ALD- (term2) z1] x2 -3' and 5 '- [(prom3) _y2 -NOXE- (term3) _z2 ] _x6 -3 ';
(IId) 5 '- [( prom5) y1 -ALS-term5] x5 - [prom1-ALS-term1] x1 - [prom2-ALD- (term2) z1] x2 -3' and 5 '- [(prom3) _y2 -NOXE- (term3) _z2 ] _x7 -3 '
(Where:
-ALS, ALD, NOXE; “prom1,” “prom2,” “prom3,” “prom5,” “term1,” “term2,” “term3” and “term5”; “x1”, “x2” and “x3” And `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '' are, for example, those defined above;
-`` X1 '' to `` x3 '', `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '' are the same or different for each formula (IIc) and (IId); and
-"X6" and "x7" are in the range 0 to 50, preferably 0 to 20, preferably 0 to 12, more particularly 2 to 5, preferably 3 to 4, and more preferably still Represents an integer equal to 3, with one of `` x6 '' and `` x7 '' representing 0)
Of DNA constructs may be included.

  The invention also belongs to the use of the recombinant yeast according to the invention for the production of acetoin and / or its derivatives, including methyl vinyl ketone.

The invention also relates to a method for the production of acetoin, said method comprising the following steps:
(a) culturing the recombinant yeast according to the invention in a suitable culture medium; and
(c) including a step of recovering acetoin.

  Preferably, the culture medium comprises a carbon source preferably selected from the group comprising glucose and sucrose.

FIG. 3 shows metabolic pathways in recombinant yeast strains that advantageously replace ethanol production with acetoin.

Definitions As used herein, the term “microorganism” refers to a yeast that has not been artificially modified. A microorganism can be a “donor” when it is provided with an exogenous genetic factor that is incorporated into and expressed by a microorganism “acceptor” or as a tool for gene construction or protein expression. The microorganism of the present invention is selected from yeasts that express a gene for acetoin biosynthesis.

  As used herein, the term “recombinant microorganism” or “genetically modified microorganism” or “recombinant yeast” or “genetically modified yeast” refers to a genetically modified or genetically modified yeast. . This is in accordance with the normal meaning of these terms that the microorganism of the present invention is not found in nature and is modified either by introduction or deletion or modification of genetic factors from the corresponding microorganism found in nature. means. It can also be modified by forcing the development and evolution of new metabolic pathways by combining directed mutagenesis and evolution under certain selection pressures (see, eg, WO 2004/076659) .

  When exogenous genes are introduced into a microorganism with all elements that allow expression in the host microorganism, the microorganism can be modified to express these genes. The microorganism may be modified to regulate the expression level of the endogenous gene. Modification or “transformation” of exogenous DNA in microorganisms such as yeast is a routine task for those skilled in the art. In particular, genetic modifications of microorganisms according to the present invention, and more particularly the genetic modifications defined herein, are described in DiCarlo et al. (Nucl. Acids Res., Vol. 41, No. 7, 2013: 4336. -4343) may be performed by using the CRISPR-Cas system as described.

  The term “endogenous gene” means that the gene was present in the microorganism in the wild type strain prior to any genetic modification. Endogenous genes are overexpressed by introducing heterologous sequences in addition to or replacing endogenous regulatory elements, or by introducing one or more supplemental copies of genes into a chromosome or plasmid. May be. Endogenous genes may be modified to regulate their expression and / or activity. For example, mutations may be introduced into the coding sequence to alter the gene product, or heterologous sequences may be introduced in addition to or replacing the endogenous regulatory element. Regulation of an endogenous gene can result in up-regulation and / or enhancement of the activity of the gene product, or down-regulation and / or attenuation of the activity of the endogenous gene product. Another way to enhance expression of an endogenous gene is to introduce one or more supplemental copies of the gene into a chromosome or plasmid.

  The term “exogenous gene” means that the gene has been introduced into the microorganism by means well known by those skilled in the art, while the gene is not naturally present in the wild-type microorganism. Microorganisms can express these genes when the exogenous genes are introduced into the microorganism along with all the elements that allow their expression in the host microorganism. It is routine for those skilled in the art to transform microorganisms with exogenous DNA. The exogenous gene may be integrated into the host chromosome or expressed exogenously from a plasmid or vector. Various plasmids that differ with respect to their origin of replication in cells and their copy number are all known in the art. The sequence of the exogenous gene may be adapted for its expression in the host microorganism. Indeed, those skilled in the art know about the idea of codon usage bias and how to adapt the nucleic sequence to a particular codon usage bias without altering the putative protein.

  The term “heterologous gene” means that the gene is derived from a species of microorganism that is different from the recipient microorganism that expresses it. The term refers to a gene that does not naturally occur in a microorganism.

  In this application, all genes are referenced with reference to their common names as well as their nucleotide sequences and in their cases their amino acid sequences. Using the reference given to the accession number for a known gene, one skilled in the art can determine the corresponding gene in other organisms, bacterial strains, yeast, fungi, mammals, plants and the like. This routine work uses consensus sequences that can be determined by aligning sequences with genes from other microorganisms and designing degenerate probes to clone the corresponding genes in another organism. Advantageously.

  The person skilled in the art knows different means of regulating the expression of endogenous genes and in particular upregulating or downregulating them. For example, another way to enhance expression of an endogenous gene is to introduce one or more supplemental copies of the gene into a chromosome or plasmid.

  Another approach is to replace the gene's endogenous promoter with a stronger promoter. These promoters may be homologous or heterologous. Homologous promoters known to allow high levels of expression in yeast are those selected in groups such as ADH1, GPDH, TEF1, truncated HXT7, PFK1, FBA1, PGK1 and TDH3. Particularly interesting promoters in the present invention are defined in more detail below.

  In yeast, the nucleic acid expression construct is preferably operably linked to a nucleic acid sequence encoding each of the genes considered and, more particularly, each of the aforementioned ALS, ALD and NOXE enzymes according to the present invention. Regulatory sequences, eg, promoter and terminator sequences.

  The nucleic acid expression construct may further comprise a 5 ′ and / or 3 ′ recognition sequence and / or a selectable marker.

  The term “overexpression” means that the expression of a gene or enzyme is increased compared to an unmodified microorganism. Increased expression of the enzyme can be obtained by increasing the expression of the gene encoding the enzyme. Increasing gene expression may be done by any technique known by those skilled in the art. In this regard, mention may be made in particular of the implementation of a strong promoter upstream of a nucleic acid intended to overexpress or introduce several copies of said nucleic acid between a promoter, in particular a strong promoter and terminator.

  Enzyme “activity” is used interchangeably with the term “function” and, in the context of the present invention, refers to the ability of an enzyme to catalyze a desired reaction.

  The terms “reduced activity” or “attenuated activity” of an enzyme refer to a reduced specific catalytic activity of a protein resulting from a mutation in the amino acid sequence and / or a deletion of the corresponding gene in the cognate due to a mutation in the nucleotide sequence. Means any of the reduced concentrations of the protein in the cells obtained.

  The term “enhanced activity” of an enzyme refers to, for example, the increased specific catalytic activity of the enzyme and / or the increased amount / availability of the enzyme in the cell, obtained by overexpression of the gene encoding the enzyme. Specify one.

  The term “encoding” or “coding” refers to the process by which a polynucleotide produces an amino acid sequence through transcriptional and translational mechanisms.

  The gene encoding the enzyme contemplated in the present invention can be exogenous or endogenous.

  “Attenuation” of a gene means that the gene is expressed at a lower rate than in an unmodified microorganism. Attenuation may be achieved by means and methods known by those skilled in the art, such as gene deletion obtained by homologous recombination, gene attenuation by insertion of an external element into the gene or under a weak promoter. Contains gene expression. Those skilled in the art are aware of various promoters that exhibit different strengths and promoters used for weak genetic expression.

  The method practiced in the present invention is preferably a functional disruption of one or more target genes for the stable introduction of heterologous nucleotide sequences at specific positions in the chromosome or in genetically modified microbial cells. Requires the use of one or more chromosomal integration constructs for In some embodiments, disruption of the target gene prevents expression of the relevant functional protein. In some embodiments, disruption of the target gene results in expression of a non-functional protein from the disrupted gene.

  Parameters for chromosomal integration constructs that may vary in the practice of the invention include homologous sequence length; homologous sequence nucleotide sequence; integration sequence length; integration sequence nucleotide sequence; and target locus nucleotide sequence. However, it is not limited to these. In some embodiments, the effective range for the length of each homologous sequence is 20 to 5,000 base pairs, preferentially 50 to 100 base pairs. In certain embodiments, the length of each homologous sequence is about 50 base pairs. For more information on the length of homology required for gene targeting, see D. Burke et al., Methods in yeast Genetics-A cold spring harbor laboratory course Manual (2000).

  In some embodiments, the disrupted pyruvate decarboxylase gene, into which the DNA construct described above is intended to be inserted, is one or more selectable useful for selection of transformed microbial cells. Markers may be advantageously included. Preferably, the selectable marker is included in a DNA construct according to the present invention.

  In some embodiments, the selectable marker is an antibiotic resistance marker. Examples of antibiotic resistance markers include, but are not limited to, NAT1, AURl-C, HPH, DSDA, KAN <R>, and SH BLE gene products. NAT1 gene product from S. noursei confers resistance to nourseothricin; AURl-C gene product from budding yeast confers resistance to aureobasidin A (AbA); pneumonia Klebsiella pneumonia HPH gene product confers resistance to hygromycin B; E. coli DSDA gene product allows cells to grow on plates with D-serine as the sole nitrogen source The KAN <R> gene of the Tn903 transposon confers resistance to G418; the SH BLE gene product from Streptoalloteichus hindustanus confers resistance to Zeocin (bleomycin).

  In some embodiments, the antibiotic resistance marker is deleted after the genetically modified microbial cell of the invention is isolated. One skilled in the art can select an appropriate marker for a particular gene.

  In some embodiments, the selectable marker rescues auxotrophy (eg, auxotrophy) in the genetically modified microbial cell. In such embodiments, the parental microbial cell is in an amino acid or nucleotide biosynthetic pathway in yeast that renders the parental microbial cell incapable of growing in media that is not supplemented with one or more nutrients (auxotrophic phenotype). Includes functional disruption in one or more functioning gene products, eg, HIS3, LEU2, LYS1, LYS2, MET15, TRP1, ADE2, and URA3 gene products. Second, the auxotrophic phenotype is the function of the disrupted gene product (NB: a functional copy of the gene can be from a close species such as Kluveromyces, Candida). Can be rescued by transforming the parental microbial cell with a chromosomal integrant that encodes the genetic copy, and the resulting genetically modified microbial cell is deficient in the auxotrophic phenotype of the parental microbial cell. Can be selected based on.

  Reference sequences are described herein for each of the nucleic acid sequences including promoter sequences, coding sequences (eg, enzyme coding sequences), or terminator sequences. The description also encompasses nucleic acid sequences having a particular percentage nucleic acid identity with the reference nucleic acid sequence.

  For each amino acid sequence of interest, a reference sequence is described herein. The description also encompasses amino acid sequences (eg, enzyme amino acid sequences) having a particular amino acid sequence identity (%) with a reference amino acid sequence.

  For obvious reasons, in all this description, a specific nucleic acid sequence or a specific amino acid sequence, according to the nucleotide or amino acid identity considered, respectively, will yield a protein (or enzyme) that exhibits the desired biological activity. Should be further guided. As used herein, ``% identity '' between two nucleic acid sequences or between two amino acid sequences is determined by comparing both sequences that are optimally aligned through a comparison window. The

  Thus, a portion of the nucleotide or amino acid sequence in the comparison window adds or deletes (e.g., `` gap '') (these additions or these deletions) so as to obtain an optimal alignment between both sequences. (Not included) may be included relative to the reference sequence.

  The percent identity determines the number of positions that can be found for both sequences to which the same nucleobase or the same amino acid residue is compared, and then identity is between both nucleobases or both amino acids. Divide the number of positions that can be observed between the residues by the total number of positions in the comparison window, then multiply the result by 100 to determine the percent nucleotide identity between the two sequences or the two sequences. Calculated by obtaining the percent amino acid identity between.

  The comparison of the optimal alignment of the sequences may be performed by a computer using a known algorithm.

  Most preferably, the percent sequence identity is determined using CLUSTAL W software (version 1.82) with the parameters set as follows: (1) CPU MODE = ClustalW mp; (2) ALIGNMENT = "full "; (3) OUTPUT FORMAT =" aln w / numbers "; (4) OUTPUT ORDER =" aligned "; (5) COLOR ALIGNMENT =" no "; (6) KTUP (word size) =" default "; (7 ) WINDOW LENGTH = "default"; (8) SCORE TYPE = "percent"; (9) TOPDIAG = "default"; (10) PAIRGAP = "default"; (11) PHYLOGENETIC TREE / TREE TYPE = "none"; ( 12) MATRIX = "default"; (13) GAP OPEN = "default"; (14) END GAPS = "default"; (15) GAP EXTENSION = "default"; (16) GAP DISTANCES = "default"; (17 ) TREE TYPE = "cladogram" and (18) TREE GRAP DISTANCES = "hide".

  "Fermentation" or "culture" is generally performed in a fermentor with a suitable culture medium that is compatible with the microorganism being cultured and contains at least one simple carbon source and optionally a co-substrate.

  The microorganisms disclosed herein may be grown in a fermentation medium to produce a product from pyruvate. For maximum production of acetoin, microbial strains used as production hosts preferably have a high proportion of carbohydrate utilization. These features may be conferred by mutagenesis and selection, genetic engineering, or may be natural. The fermentation medium or “culture medium” for the cells may contain at least about 10 g / L glucose. Additional carbon substrates include monosaccharides such as fructose, mannose, xylose and arabinose; oligosaccharides such as lactose, maltose, galactose, or sucrose; polysaccharides such as starch or cellulose or mixtures thereof and recoverable Non-refined mixtures from feedstocks such as cheese whey penetrating corn steep liquor, sugar beet molasses, and barley malt may be included, but are not limited to these. Other carbon substrates may include glycerol.

  Therefore, it is intended that the source of carbon utilized in the present invention can include a variety of carbon containing substrates and is limited only by the choice of organism.

  Although all the above carbon substrates and mixtures thereof are intended to be suitable for the present invention, preferred carbon substrates are those of microorganisms modified to use glucose, fructose, and sucrose, or these and C5 sugars. For C5 sugars, such as mixtures with xylose and / or arabinose, and more particularly glucose.

  A preferred carbon substrate is sucrose.

  According to a particular embodiment, the carbon substrate according to the invention does not consist of xylose.

  In addition to a suitable carbon source, the fermentation medium is known to those skilled in the art, suitable minerals, salts, cofactors, suitable for promoting the enzymatic pathway required for growth of the culture and production of the desired product, Buffering agents and other ingredients may be included.

  In addition, additional genetic modifications suitable for the growth of recombinant microorganisms according to the present invention may be considered.

  In order to prevent acetoin escape to 2,3-BDO, genes involved in the reduction of acetoin to 2,3-BDO may be inactivated. In this regard, endogenous butanediol dehydrogenase bdh1 and bdh2 (particularly known by EC number 1.1.1.4) can be cited, but endogenous alcohol dehydrogenases adh1, adh3 and adh4 (particularly known by ECC number 1.1.1.1). Can also be cited. The inactivation of the gene belongs to the technical common sense of those skilled in the art.

  The presence of weak acids is known to be limited for growth and is often present in cellulose or molasses-derived media.

  Additional genetic modifications such as disruption of the JEN1 gene (or strain name: YKL217W or protein accession number P36035 (UniProtKB swiss-Prot)) and / or HAA-1 gene (strain name: YPR008W or accession number Q12753 (UniProtKB Overexpression of swiss-Prot)) leads to improvements in strains resistant to weak acids in the culture medium in effect.

  Jen1 is a membrane protein responsible for lactic acid uptake and transport in cells (Casal M, et al. (1999), J. Bacteriol., 181 (8): 2620-3).

  HAA-1 is a transcriptional activator that regulates the expression of membrane stress proteins that bear resistance to weak acids. Its overexpression enhances yeast resistance to acetic acid (Tanaka et al. (2012) Appl Environ Microbiol., 78 (22): 8161-3).

  Disruption of the JEN1 gene and overexpression of the HAA-1 gene belong to the common general technical knowledge of those skilled in the art, and may be particularly performed using the methods shown herein.

  In view of the following formula for the synthesis of acetoin in yeast, the conditions considered in the present invention are necessarily aerobic conditions.

  The term “aerobic condition” refers to the concentration of oxygen in the culture medium that is sufficient for aerobic or facultative anaerobic microorganisms that use dioxygen as the final electron acceptor.

“Microaerobic conditions” refers to a culture medium in which the concentration of oxygen is less than that in air, ie up to 6% O ₂ .

  “Suitable culture medium” includes, for example, a carbon source or substrate, a nitrogen source such as peptone, yeast extract, meat extract, malt extract, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; E.g. monopotassium phosphate or dipotassium phosphate; trace elements (e.g. metal salts), e.g. magnesium salts, cobalt salts and / or manganese salts; and cells such as growth factors e.g. amino acids, vitamins, growth promoters Specify a medium (eg, a sterile liquid medium) that contains nutrients essential or beneficial for maintenance and / or growth. The terms `` carbon source '' or `` carbon substrate '' or `` carbon source '' according to the present invention are hexoses (e.g. glucose, galactose or lactose), pentoses, monosaccharides, oligosaccharides, disaccharides (e.g. sucrose, cellobiose). Or maltose), any source of carbon that can be used by those skilled in the art to support the normal growth of microorganisms, including molasses, starch or derivatives thereof, cellulose, hemicellulose and combinations thereof.

Recombinant yeast according to the invention As mentioned above, the present invention relates to a recombinant yeast having reduced pyruvate decarboxylase activity, the following being inserted into its genome:
-One or more nucleic acids encoding acetolactate synthase or ALS,
-One or more nucleic acids encoding acetolactate decarboxylase or ALD, and
-One or more copies of nucleic acid encoding NADH oxidase (or NOXE).

  As shown in the Examples herein, the inventors have further identified genes that have reduced pyruvate decarboxylase activity and allow expression of ALS and ALD enzymes required for acetoin synthesis. In the integrated recombinant yeast, the presence of a nucleic acid encoding NADH oxidase, preferably the presence of multiple copies thereof, not only contributes to the stabilization of said recombinant yeast, but also the growth of this strain and the production of acetoin. It was unexpectedly found that a significant increase in the yield of can also be achieved.

  The use of Crabtree positive yeast, such as Saccharomyces cerevisiae, in particular recombinant yeasts, such as Saccharomyces cerevisiae, for the production of metabolites of interest is advantageous because yeast cells, in contrast to bacteria, This is because it has the ability to perform fermentation in the presence of oxygen in the presence of a sufficient amount of sugar such as glucose or sucrose. In contrast, bacteria undergo fermentation only under anaerobic conditions. In addition, yeast is not virally infected, as opposed to having bacteriophages for bacteria. Still further, yeast cultures are rarely contaminated by undesired microorganisms such as bacteria, because the yeast cells are in the environment, for example, the rapid growth of culture media up to pH 4 to support growth. This is because it causes acidification. Still further, yeast cells do not excrete some unwanted metabolites, such as lactic acid, whose presence in the culture medium is a practical weakness for subsequent purification of the metabolite of interest. Still further, yeast, including recombinant yeast, has a high degree of genetic stability compared to bacteria.

  The formula for the synthesis of acetoin in yeast is the following:

  Said mass formula may be in accordance with the fact that budding yeast can be fermented even in the presence of oxygen.

  In view of the above formula, the maximum theoretical yield of acetoin would be 97.78 g for an input of 200 g glucose.

  As shown in the examples herein, the effective yield of acetoin in the recombinant yeast according to the present invention is relatively close to this maximum theoretical yield (up to 83%). According to the knowledge of the inventor, such a yield has never been obtained in yeast so far.

  Thus, production of acetoin in high yield has been successfully achieved in the recombinant yeast according to the present invention, and the path for industrial production of acetoin in yeast is in place.

  Surprisingly, as also shown in the examples herein, non-toxicity of acetoin produced in yeast cells is observed even at high concentrations of synthesized acetoin. Moreover, the purified process is substantially simplified because the synthesized acetoin is completely excreted and transported outside the cell.

The NADH oxidase (or NOXE) used in the recombinant yeast according to the invention is a very specific “NADH-dependent” enzyme and does not consume any carbonate acceptor. For this reason, the selected NADH oxidase does not directly interfere with carbonic acid metabolism, but replenishes the water produced with a NAD ⁺ pool.

  In this regard, the NADH oxidase used in the recombinant yeast according to the present invention is the “NADH-dependent” disclosed in the prior art documents mentioned above and in particular in US patent application No. 2011/0124060 and WO 2013/076144. “It is significantly different from the enzyme.

According to a particular embodiment, the recombinant yeast has the formula:
(I) 5 '-[gene 1] _x1 -3' and 5 '-[gene 2] _x2 -3' and 5 '-[gene 3] _x3 -3',
(II) 5 '-[gene 1] _x1- [gene 2] _x2 -3' and 5 '-[gene 3] _x3 -3',
(III) 5 '-[gene 1] _x1- [gene 2] _x2- [gene 3] _x3 -3', and combinations thereof,
(Where:
-"Gene 1" means a nucleic acid selected from the group comprising ALS, ALD or NOXE;
-"Gene 2" means a nucleic acid selected from the group comprising ALS, ALD or NOXE, but different from gene 1;
-"Gene 3" means a nucleic acid selected from the group comprising ALS, ALD or NOXE, but different from genes 1 and 2;
-"ALS" is a nucleic acid encoding acetolactate synthase;
-"ALD" is a nucleic acid encoding acetolactate decarboxylase;
-"NOXE" is a nucleic acid encoding NADH oxidase;
-Each of "x1", "x2" and "x3", independently of one another, represents an integer in the range of 0 to 50, preferably 0 to 20,
However, the recombinant yeast contains at least one nucleic acid encoding each of ALS, ALD and NOXE)
One or more DNA constructs selected from the group comprising:

  Preferably, each of “x1”, “x2” and “x3” independently of each other represents an integer in the range 0 to 10, more particularly in the range 0 to 5, in particular in the range 0 to 3. And better represents an integer equal to 1.

  As intended herein, each of x1, x2, and x3 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 and 50.

  In the DNA constructs of the above formulas (I) to (III), one or more integers `` x1 '', `` x2 '' and / or `` x3 '', independently of each other, have a value of 2 or more. In form, each of the two or more copies of the corresponding gene 1, gene 2 and / or gene 3 of the related genes may be the same or different. Various distinct sequences of ALS, ALD and NOXE are shown in Table 1 herein.

  Exemplary embodiment of a DNA construct selected from those of formulas (I) to (III) above, wherein `` x1 '' is an integer equal to 2 and gene 1 is a nucleic acid encoding acetolactate synthase (ALS) Then, the two ALS coding sequences contained in the DNA construct may be the same or different.

  For example, according to this particular embodiment, this is that the first copy of the nucleic acid encoding acetolactate synthase may be a nucleic acid encoding ALS.Bs and the second of the nucleic acid encoding acetolactate synthase. This means that a nucleic acid encoding ALS.Pp may be used.

  In an embodiment of the recombinant yeast according to the invention, wherein said recombinant yeast comprises at least two DNA constructs selected from the group comprising DNA constructs of formulas (I) to (III), each DNA construct is more detailed In addition, each of the related genes 1, 2 and / or 3 contained therein may be the same or different.

  In the following, some exemplary embodiments of DNA constructs selected from the group comprising DNA constructs of formula (I), (II) and (III) are presented.

Recombinant yeast comprising one DNA construct of formula (I):
(I) 5 '-[ALS] ₂ -3' and 5 '-[ALD] ₂ -3' and 5 '-[NOXE] ₃ -3'
A recombinant yeast comprising a DNA construct of formula (I) above has reduced pyruvate decarboxylase activity and is introduced into its genome with the following three DNA partial constructs (i) to (iii):
(i) DNA partial constructs that are the same or different from each other and comprise two nucleic acids, each nucleic acid encoding ALS, wherein the DNA partial construct is introduced at a first position in the genome of the recombinant yeast; DNA partial constructs;
(ii) DNA partial constructs identical or different from each other and comprising two nucleic acids, each nucleic acid encoding ALD, said DNA partial construct being introduced at a second position in said recombinant yeast genome, A DNA partial construct, wherein the position is distinct from the position at which the DNA partial construct comprising the nucleic acid encoding ALS is inserted; and
(iii) DNA partial constructs that are the same or different from each other and include three nucleic acids, each nucleic acid encoding NOXE, wherein the DNA partial construct is introduced at a third position in the genome of the recombinant yeast, The position has a DNA partial construct that is distinct from the first and second positions, respectively, into which a DNA partial construct comprising a nucleic acid encoding ALS and a DNA partial construct comprising a nucleic acid encoding ALD are inserted. .

  In some embodiments, the required reduced pyruvate decarboxylase activity of the particular recombinant yeast is at least one of at least one DNA partial construct (i) to (iii), or alternatively a combination thereof. It can be obtained by insertion in one yeast pdc gene.

Recombinant yeast comprising one DNA construct of formula (II):
(II) 5 '-[ALS] _2- [ALD] ₂ -3' and 5 '-[NOXE] ₃ -3'
A recombinant yeast comprising a DNA construct of formula (II) above has a reduced pyruvate decarboxylase activity and has a genome into which the following two DNA partial constructs (A) and (B) are inserted:
(A) a first DNA partial construct 5 ′-[ALS] _2- [ALD] ₂ -3 ′, introduced at the first position in the genome of the recombinant yeast, the following:
(i) two nucleic acids that are the same or different from each other, each nucleic acid encoding ALS; and
(ii) the first DNA partial construct comprising two nucleic acids, identical or different from each other, each nucleic acid encoding ALD; and
(B) a second DNA partial construct 5 ′-[NOXE] ₃ -3 ′, which is introduced at the second position in the genome of the recombinant yeast, where the first DNA partial construct is inserted. Said second partial DNA construct, wherein said second partial DNA construct is separated from said first position and comprises (iii) the same or different from each other, each comprising three nucleic acids encoding NOXE.

  In certain embodiments, the required reduced pyruvate decarboxylase activity of the particular recombinant yeast is in at least one yeast pdc gene of the first DNA partial construct and / or the second DNA partial construct. Can be obtained by insertion.

Recombinant yeast comprising one DNA construct of formula (III):
(III) 5 '-[ALS] _2- [ALD] _2- [NOXE] ₃ -3'
A recombinant yeast comprising the DNA construct of formula (III) above has a reduced pyruvate decarboxylase activity and a genome into which one DNA construct located at a desired position in the genome of the recombinant yeast is inserted And the DNA construct is:
(i) two nucleic acids that are the same or different from each other, each nucleic acid encoding ALS;
(ii) two nucleic acids that are the same or different from each other, each nucleic acid encoding ALD; and
(iii) includes three nucleic acids that are identical or different from each other, each nucleic acid encoding NOXE.

  In certain embodiments, the required reduced pyruvate decarboxylase activity of the particular recombinant yeast can be obtained by insertion of the DNA construct in at least one yeast pdc gene.

For each of these three exemplary embodiments of the recombinant yeast according to the present invention, as described above, `` x1 '' to `` x3 '', independently of each other, represent integers having a value of 2 or more:
-One copy of ALS in a single DNA construct may be identical to another copy of ALS contained in said DNA construct or identical to all other copies of ALS contained in said DNA construct. Alternatively, the one copy ALS may alternatively be separate from each other's copy ALS contained in the DNA construct.
-One copy of ALD in a single DNA construct may be identical to another copy of ALD contained in said DNA construct or identical to all other copies of ALD contained in said DNA construct. Alternatively, the one copy ALD may alternatively be separate from each other's copy ALD contained in the DNA construct.
-One copy of NOXE within a single DNA construct may be identical to another copy of NOXE contained in said DNA construct or identical to all other copies of NOXE contained in said DNA construct. Alternatively, the one copy of NOXE may alternatively be separate from each other copy of NOXE contained in the DNA construct.

Recombinant yeast comprising one DNA construct of formula (III) and one DNA construct of formula (I):
(III) 5 '-[ALS] _1- [ALD] _1- [NOXE] ₁ -3', and
(I) 5 '-[ALS] ₀ -3' and 5 '-[ALD] ₀ -3' and 5 '-[NOXE] ₁₂ -3'
The resulting recombinant yeast has reduced pyruvate decarboxylase activity and has a genome into which the following two DNA partial constructs (A) and (B) are inserted:
(A) the first DNA partial construct 5 ′-[ALS] _1- [ALD] _1- [NOXE] ₁ -3 ′, introduced at the first position in the genome of the recombinant yeast, thing:
(i) one nucleic acid encoding ALS;
(ii) one nucleic acid encoding ALD; and
(iii) the first partial DNA construct comprising one nucleic acid encoding NOXE;
And
(B) second DNA partial constructs 5 ′-[ALS] ₀ -3 ′ and 5 ′-[ALD] ₀ -3 ′ and 5 ′-[NOXE] ₁₂ -3 ′, wherein the recombinant yeast Introduced at a second position in the genome, the following:
(i) The second DNA partial construct comprising 12 nucleic acids encoding NOXE.

Recombinant yeast comprising two DNA constructs of formula (III) and one DNA construct of formula (I):
(III-1) 5 '-[ALS] _1- [ALD] _1- [NOXE] ₁ -3',
(III-2) 5 '-[ALS] _1- [ALD] _1- [NOXE] ₁ -3',
(I) 5 '-[ALS] ₀ -3' and 5 '-[ALD] ₀ -3' and 5 '-[NOXE] ₁₂ -3'
The resulting recombinant yeast has reduced pyruvate decarboxylase activity and has a genome into which the following three DNA partial constructs (A), (B) and (C) are inserted:
(A) the first DNA partial construct 5 ′-[ALS] _1- [ALD] _1- [NOXE] ₁ -3 ′, introduced at the first position in the genome of the recombinant yeast, thing:
(i) one nucleic acid encoding ALS;
(ii) one nucleic acid encoding ALD; and
(iii) the first partial DNA construct comprising one nucleic acid encoding NOXE;
(B) a second DNA partial construct 5 ′-[ALS] _1- [ALD] _1- [NOXE] ₁ -3 ′, introduced at a second position in the genome of the recombinant yeast, thing:
(i) one nucleic acid encoding ALS;
(ii) one nucleic acid encoding ALD; and
(iii) the second DNA partial construct comprising one nucleic acid encoding NOXE;
And
(C) third DNA partial constructs 5 ′-[ALS] ₀ -3 ′ and 5 ′-[ALD] ₀ -3 ′ and 5 ′-[NOXE] ₁₂ -3 ′, Introduced at a third position in the genome, the following:
(i) The third DNA partial construct comprising 12 nucleic acids encoding NOXE.

Recombinant yeast comprising two DNA constructs of formula (II) and one DNA construct of formula (I):
(II-1) 5 '-[ALS] _1- [ALD] ₁ -3' and 5 '-[NOXE] ₀ -3',
(II-2) 5 '-[ALS] _1- [ALD] ₁ -3' and 5 '-[NOXE] ₀ -3',
(I) 5 '-[ALS] ₀ -3' and 5 '-[ALD] ₀ -3' and 5 '-[NOXE] ₁₂ -3'

The resulting recombinant yeast has reduced pyruvate decarboxylase activity and has a genome into which the following three DNA partial constructs (A), (B) and (C) are inserted:
(A) First DNA partial constructs 5 ′-[ALS] _1- [ALD] ₁ -3 ′ and 5 ′-[NOXE] ₀ -3 ′, the first position in the genome of the recombinant yeast Introduced into the following:
(i) one nucleic acid encoding ALS; and
(ii) the first DNA partial construct comprising one nucleic acid encoding ALD;
(B) Second DNA partial constructs 5 ′-[ALS] _1- [ALD] ₁ -3 ′ and 5 ′-[NOXE] ₀ -3 ′, the second position in the genome of the recombinant yeast Introduced into the following:
(i) one nucleic acid encoding ALS; and
(ii) the second partial DNA construct comprising one nucleic acid encoding ALD;
And
(C) Third DNA partial construct 5 ′-[NOXE] ₁₂ -3 ′, which is introduced at the third position in the genome of the recombinant yeast, where the first DNA partial construct is inserted. (Iii) a partial DNA construct comprising 12 nucleic acids, which are remote from the first position, and are (iii) identical or different from each other, each encoding NOXE.

  In certain embodiments, the required reduced pyruvate decarboxylase activity of the particular recombinant yeast is in at least one yeast pdc gene of the first DNA partial construct and / or the second DNA partial construct. Can be obtained by insertion.

  According to a particular embodiment, the recombinant yeast according to the invention is at least one, preferably at least two DNA constructs of formula (II) above, wherein “gene 3” is NADH oxidase (or NOXE). It may contain a DNA construct that signifies the encoding nucleic acid.

  According to these particular embodiments, each nucleic acid of gene 1 and gene 2 necessarily means a nucleic acid selected from the group comprising ALS and ALD. In these embodiments, there are at least one copy of ALS and ALD inserted. In embodiments where only one construct of formula (II) is inserted into the yeast genome, each nucleic acid of gene 1 and gene 2 necessarily means a nucleic acid selected from the group comprising ALS and ALD, and ALS And one copy of each ALD. In embodiments where a set of two or more constructs of formula (II) is inserted into the yeast genome, each nucleic acid of gene 1 and gene 2 necessarily means a nucleic acid selected from the group comprising ALS and ALD. , At least one copy of each of ALS and ALD is present in the set of two or more constructs of formula (II).

  In addition, when the recombinant yeast according to the present invention comprises at least two DNA constructs of the above formula (II), the above DNA constructs of the formula (II) may be the same or different. .

According to a preferred embodiment, the recombinant yeast according to the invention is the same or different, at least one, preferably at least two DNA constructs of formula (IIa), each formula (IIa) having the following formula:
(IIa) 5 '- [( prom5) y1 - Gene 1-term5] _x5 - [prom1- gene 1-term1] _x1 - [prom2- gene _{2- (term2) z1] x2 -3} ' and 5 '- [( prom3) _y2 -gene 3- (term3) _z2 ] _x3 -3 '
(Where:
-Gene 1, gene 2 and gene 3, `` x1 '', `` x2 '' and `` x3 '' are, for example, those defined above;
-"X5" represents an integer equal to 0 or 1;
-`` Y1 '', `` y2 '', `` z1 '' and `` z2 '' represent, independently of one another, an integer equal to 0 or 1;
-When said recombinant yeast comprises at least two DNA constructs of formula (IIa), `` x1 '', `` x2 '', `` x3 '', `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '''' May be the same or different;
-"Prom 1" is a regulatory sequence that controls the expression of the sequence encoding gene 1;
-"Prom 2" is a regulatory sequence that controls the expression of the sequence encoding gene 2;
-"Prom 3" is a regulatory sequence that controls the expression of the sequence encoding gene 3;
-`` Prom 5 '' is a control sequence that controls the expression of gene 1, said prom 5 being the same as or different from prom 1;
-"Term 1" is a transcription terminator sequence that terminates the expression of the sequence encoding gene 1;
-"Term 2" is a transcription terminator sequence that terminates the expression of the sequence encoding gene 2;
-"Term 3" is a transcription terminator sequence that terminates the expression of the sequence encoding gene 3; and
-"Term 5" is a transcription terminator sequence that terminates the expression of gene 1, and said term 5 is the same as or different from term 1)
A DNA construct having

In order to make clearer with respect to the features `` x5 '' and `` y1 '', examples are given below that illustrate in more detail specific related embodiments:
If "x5" is an integer equal to 1 and "y1" represents an integer equal to 0, the gene 1 considered is the promoter control of the recombinant yeast gene into which the DNA construct to be considered is inserted Means that if x5 is an integer equal to 1 and y1 represents an integer equal to 1, the gene 1 being considered is under the control of the promoter “prom5” Means. In this regard, the promoter sequence of the endogenous gene, preferably the pdc gene, into which the DNA construct is inserted is excluded or at least interrupted, as well as the sequence of its associated coding region.

In addition, especially with respect to the features `` y2 '' and `` z2 '', examples are given below that illustrate in more detail certain related embodiments (of course, in these following examples, `` x3 '' (Represents an integer greater than 1):
If `` y2 '' is an integer equal to 0, it means that the gene 3 considered is under the control of the promoter of the recombinant yeast gene into which the DNA construct considered is inserted; or If y2 "is an integer equal to 1, it means that the gene 3 considered is under the control of the promoter" prom3 ". In this regard, the promoter sequence of the endogenous gene into which the DNA construct is inserted is the sequence of the coding region that is excluded or at least interrupted, as well as its associated.
If “z2” is an integer equal to 0, this means that the gene 3 considered is linked to the transcription terminator of the gene of the recombinant yeast into which the DNA construct to be considered is inserted; or If z2 "is an integer equal to 1, it means that the gene 3 considered is linked to the transcription terminator" term3 ". In this regard, the sequence of the transcription terminator of the endogenous gene into which the DNA construct is inserted is eliminated or at least interrupted, as well as the sequence of its associated coding region.

  With respect to “z1”, where present in the formulas described herein, the above for “z2” applies mutatis mutandis.

According to another preferred embodiment, the recombinant yeast according to the invention may comprise at least one, preferably at least two DNA constructs of the following formula (IIb):
(IIb) 5 '- [( prom5) y1 -ALS-term5] x5 - [prom1-ALS-term1] x1 - [prom2-ALD- (term2) z1] x2 -3' and 5 '- [(prom3) _y2 -NOXE- (term3) _z2 ] _x3 -3 '
(Where:
-ALS, ALD, NOXE, `` x1 '', `` x2 '', `` x3 '', `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '' are, for example, those defined above;
-If said recombinant yeast comprises at least two DNA constructs of formula (IIb), `` x1 '', `` x2 '', `` x3 '', `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '' May be the same or different;
-"Prom 1" is a regulatory sequence that controls the expression of the sequence encoding acetolactate synthase;
-"Prom 2" is a regulatory sequence that controls the expression of the sequence encoding acetolactate decarboxylase;
-"Prom 3" is a regulatory sequence that controls the expression of the sequence encoding NADH oxidase;
-"Prom 5" is a control sequence that controls the expression of a sequence encoding acetolactate synthase, said prom 5 being the same as or different from prom 1;
-"Term 1" is a transcription terminator sequence that terminates the expression of the sequence encoding acetolactate synthase;
-"Term 2" is a transcription terminator sequence that terminates the expression of the sequence encoding acetolactate decarboxylase;
-"Term 3" is a transcription terminator sequence that terminates the expression of the sequence encoding NADH oxidase;
“Term 5” is a transcription terminator sequence that terminates the expression of the sequence encoding acetolactate synthase, and the term 5 is the same as or different from the term 1).

  According to another preferred embodiment, the recombinant yeast according to the invention may comprise at least two DNA constructs of formula (II), (IIa) or (IIb) provided that all copies of NOXE nucleic acid are present. Is located in one of at least two DNA constructs of formula (II), (IIa) or (IIb).

According to another preferred embodiment, the recombinant yeast according to the invention may comprise at least two, preferably exactly two, DNA constructs of the following formulas (IIc) and (IId):
(IIc) 5 '- [( prom5) y1 -ALS-term5] x5 - [prom1-ALS-term1] x1 - [prom2-ALD- (term2) z1] x2 -3' and 5 '- [(prom3) _y2 -NOXE- (term3) _z2 ] _x6 -3 ';
(IId) 5 '- [( prom5) y1 -ALS-term5] x5 - [prom1-ALS-term1] x1 - [prom2-ALD - (term2) z1] x2 -3' and 5 '- [(prom3) _y2 -NOXE- (term3) _z2 ] _x7 -3 ';
(Where:
-ALS, ALD, NOXE, "prom1,""prom2,""prom3,""prom5,""term1,""term2,""term3,""term5,""x1,""x2,""x3" , “X5”, “y1”, “y2”, “z1” and “z2” are, for example, those defined above; and
-`` X1 '' to `` x3 '', `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '' are the same or different for each formula (IIc) and (IId); and
-"X6" and "x7" are integers equal to 0 to 50, preferably 0 to 20, preferably 0 to 12, more particularly in the range 2 to 5, preferably 3 to 4, and more preferably 3. Where one of “x6” and “x7” represents 0).

  Advantageously, the first gene 1 at 5′- in the DNA constructs of formulas (I) to (III) is preferably a gene represented by a nucleic acid encoding ALS, more particularly said first gene 1 is under the control of the promoter of the recombinant yeast gene into which the considered DNA construct is inserted.

  More particularly, for a DNA construct of formula (IIa), (IIb), (IIc) or (IId), preferably “x5” represents an integer equal to 1 and “y1” represents an integer equal to 0. Means.

In view of the complexity of the above described DNA constructs and DNA partial constructs according to the present invention, the following is emphasized:
-For one DNA construct of the invention, `` x1 '', `` x2 '' and / or `` x3 '' represent an integer greater than or equal to 2:
o Each copy for the relevant nucleic acid of gene 1, gene 2 and / or gene 3 may be the same or different; and / or
o The promoter and / or terminator for each copy for the relevant nucleic acid of gene 1, gene 2 and / or gene 3 may be the same or different;
If the recombinant yeast comprises at least two DNA constructs, the at least two DNA constructs may be the same or different with respect to:
(i) their general formula, wherein the DNA construct can be characterized by a formula selected from the group comprising formulas (I) to (III);
(ii) values from “x1” to “x3” and “x5” to “x7”, “y1”, “y2”, “z1” and / or “z2”;
(iii) the nature of the promoter for the same gene;
(iv) the nature of the terminator for the same gene; and / or
(v) A property of the same gene itself that ALS, ALD and NOXE may be derived from organisms belonging to different genera, especially as shown in Table 1 below.

  Methods practiced to realize DNA constructs such as those defined above belong to the common general knowledge of those skilled in the art.

  In this regard, those skilled in the art eventually made only minor changes, Shao et al. (Nucleic Acids Research, 2009, Vol. 37, No. 2: e16) and Shao et al. (Methods in Enzymology, 2012 Elsevier Inc., The method described in Vol. 517: 203, more particularly the method developed in the following examples, can be advantageously cited.

Reduced pyruvate decarboxylase activity Endogenous pyruvate decarboxylase activity in yeast converts pyruvate to acetaldehyde, which is then converted via acetate to ethanol or acetyl-CoA.

  As previously mentioned, the present invention relates to recombinant yeast having reduced pyruvate decarboxylase activity, with a specific DNA construct inserted in its genome.

  According to a particular embodiment, the recombinant yeast is characterized by the fact that one or more endogenous pyruvate decarboxylase-encoding genes can be switched off.

  The pyruvate decarboxylase activity of the recombinant yeast according to the invention may be reduced by all methods known by the person skilled in the art.

  In this regard, the pyruvate decarboxylase activity of the recombinant yeast according to the present invention is, for example, that (i) at least one gene encoding pyruvate decarboxylase is at least within the at least one gene encoding pyruvate decarboxylase. Destroying one exogenous DNA construct by insertion by methods known by those skilled in the art, (ii) mutations in regulatory regions that reduce pyruvate decarboxylase transcription, (iii) in particular, AUG to GUG (Iv) mutations in the coding sequence that alter activity, (v) mutations in the coding sequence that alter protein stability, insertions or deletions, (vi) pyruvate decarboxylase mRNA half-life by substitution May be reduced by mutations that alter

  With respect to the first option (i), the DNA construct implemented to disrupt the considered pdc gene is an exogenous DNA construct different from the DNA construct according to the invention as described above, the DNA construct according to the invention, Or they may be a combination thereof.

  Further, as described above, the DNA constructs according to the present invention of the formulas (I) and (II) are each composed of two or more DNA partial constructs.

  Thus, according to a particular method of realization, the pyruvate decarboxylase activity of the recombinant yeast according to the present invention is such that at least one gene encoding pyruvate decarboxylase is present in said gene of formula (I) and (II). It may be reduced by destroying by inserting only at least one DNA partial construct of at least one DNA construct according to the invention.

  Preferably, endogenous pyruvate decarboxylase activity may be reduced by disruption of at least one pdc gene.

  Indeed, the yeast may have one or more genes encoding pyruvate decarboylase. For example, there is one gene encoding pyruvate decarboxylase in Kluyveromyces lactis, while on the other hand, 3 of the pyruvate decarboxylase encoded by the PDC1, PDC5, and PDC6 genes in budding yeast. There are two isozymes as well as the pyruvate decarboxylase regulatory gene PDC2.

  Preferably, the recombinant yeast according to the invention, as defined below, may be a recombinant Saccharomyces genus, and preferably a recombinant budding yeast species.

  In this regard, according to a first variant, pyruvate decarboxylase activity may be reduced by disruption of at least one pdc gene, preferably at least two pdc genes, and more particularly only two pdc genes. .

  In addition, the disrupted pdc gene may be selected from the group consisting of pdc1, pdc5, pdc6 and mixtures thereof, and preferably pdc1 and pdc6.

  Preferably, when the recombinant yeast belongs to the genus Saccharomyces, the pyruvate decarboxylase activity is preferably selected from the group consisting of pdc1, pdc5, pdc6 and combinations thereof, more particularly the group consisting of pdc1 and pdc6 May be reduced by disruption of at least two pdc genes selected from

  Indeed, disruption of the three pdc genes in the genus Saccharomyces, preferably a budding yeast species, dramatically reduces strain growth, making it incompatible with any industrial application.

  According to a particular variant, only the pdc1 and pdc6 genes are disrupted and the expression of pdc5 is attenuated in Saccharomyces, preferably a budding yeast species.

  Methods practiced to attenuate the expression of specific genes belong to the common general knowledge of those skilled in the art.

  In this regard, the person skilled in the art may advantageously refer to any method known in the art.

  Advantageously, in order to attenuate the expression of pdc5, its transcription is in particular a weak promoter such as RPLA1, URA3, MET25, HIS3, TRP1, GAP1, NUP57 or TFC1, and preferably RPLA1 (= SEQ ID NO: 37). It may be placed under control.

  The methods implemented to measure the activity level of pyruvate decarboxylase belong to the common general knowledge of those skilled in the art.

  In this regard, the person skilled in the art may advantageously cite the method described in Wang et al. (Biochemistry, 2001, 40: 1755-1763).

Acetolactate synthase Acetolactate synthase (ALS) enzyme (acetohydroxyacid synthase (AHAS), α-acetohydroxyacid synthetase, α-acetohydroxyacid synthase, α-acetolactic acid synthase, α-acetolactic acid synthetase, acetohydroxyacid synthetase, Acetohydroxyacid synthase, acetolactate pyruvate lyase (carboxylated), also known as acetolactate synthetase) catalyzes the first step in the synthesis of branched chain amino acids (valine, leucine, and isoleucine) It is a protein.

  ALS is an enzyme specifically involved in chemical reactions involved in the conversion of two pyruvate molecules into acetolactate molecules and carbon dioxide. The reaction uses thiamine pyrophosphate to link two pyruvate molecules.

  The method implemented to measure the activity level of acetolactate synthase belongs to the common general knowledge of those skilled in the art.

  In this regard, those skilled in the art may advantageously cite the method described in Poulsen et al. (Eur. J. Biochem. 185, 1989: 433-439).

  A preferred acetolactate synthase in the present invention is known by EC number 2.2.1.6.

  According to a preferred embodiment, the nucleic acid encoding acetolactate synthase or ALS is preferably Bacillus subtilis, tobacco (Nicotiana tabacum), Paenibacillus polymyxa, and mixtures thereof, and Preferably, it may be a nucleic acid selected from the group comprising tobacco and Paenibacillus polymixer.

  According to a further preferred embodiment, the nucleic acid encoding acetolactate synthase or ALS is selected from the group consisting of sequences having at least 65%, preferably at least 80% nucleic acid identity with the nucleic acid sequences SEQ ID NOs: 1, 3 and 5. It may be a nucleic acid.

  As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence is at least 66%, 67%, 68%, 69%, 70%, 71% with the reference nucleic acid sequence. 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 Nucleic acid sequences having%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity are included.

  As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence is at least 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleic acid sequences with nucleotide identity.

  According to another particular embodiment, the nucleic acid encoding acetolactate synthase is an amino acid sequence selected from the group consisting of sequences having at least 65%, preferably at least 80% identity with SEQ ID NO: 2, 5, and 6. It may be a nucleic acid encoding.

  As described herein, an amino acid sequence having at least 65% amino acid sequence identity with a reference amino acid sequence is at least 66%, 67%, 68%, 69%, 70%, 71 with said reference amino acid sequence. %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, It includes amino acid sequences having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity.

  As described herein, an amino acid sequence having at least 80% nucleotide identity with a reference amino acid sequence is at least 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid sequences are included.

  As described above, the expression level of ALS in the present invention is controlled by, for example, at least one promoter and at least one terminator, which are defined in more detail below, which are 5 ′ and N, respectively, of the nucleic acid sequence encoding ALS. Present at 3 'position.

Acetolactate decarboxylase Acetolactate decarboxylase (ALD) enzyme (α-acetolactate decarboxylase, (S) -2-hydroxy-2-methyl-3-oxobutanoate carboxy-lyase, (S) -2-hydroxy- 2-methyl-3-oxobutanoate carboxy-lyase [(R) -2-acetoin-formation] or (S) -2-hydroxy-2-methyl-3-oxobutanoate carboxy-lyase [(3R) -3-hydroxybutan-2-one-formation)) is a family of lyases, specifically breaking carbon-carbon bonds and participating in butanoate metabolism and c5-branched diacid metabolism It belongs to carboxy lyase.

  ALD is an enzyme that specifically participates in chemical reactions involved in the conversion of α-acetolactic acid molecules into acetoin molecules and carbon dioxide.

  The methods implemented to measure the activity level of acetolactate decarboxylase belong to the common general knowledge of those skilled in the art.

  In this regard, those skilled in the art may advantageously cite the method described in Dulieu et al. (Enzyme and Microbial Technology 25, 1999: 537-542).

  A preferred acetolactate decarboxylase in the present invention is known by EC number 4.1.1.5.

  According to a preferred embodiment, the nucleic acid encoding acetolactate decarboxylase or ALD is Brevibacillus brevis, Enterobacter aerogenes, Lactococcus lactis, and mixtures thereof, preferably It may be a nucleic acid selected from the group comprising Brevibacillus brevis and Erobacter erogenes.

  According to a further preferred embodiment, the nucleic acid encoding acetolactate decarboxylase or ALD is selected from the group consisting of sequences having at least 36%, preferably at least 80% nucleic acid identity with the nucleic acid sequences SEQ ID NOs: 7, 9 and 11. It may be a nucleic acid.

  As described herein, a nucleic acid sequence having at least 36% nucleotide identity with a reference nucleic acid sequence is at least 37%, 38%, 39%, 40%, 41%, 42% with the reference nucleic acid sequence. , 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 %, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleic acid sequences.

  According to another particular embodiment, the nucleic acid encoding acetolactate decarboxylase is an amino acid selected from the group consisting of sequences having at least 36%, preferably at least 80% identity with SEQ ID NO: 8, 10 and 12. It may be a nucleic acid encoding a sequence.

  As described herein, an amino acid sequence having at least 36% amino acid identity with a reference amino acid sequence is at least 37%, 38%, 39%, 40%, 41%, 42% with the reference amino acid sequence. , 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 %, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid sequences are included.

  As described above, the expression level of ALD in the present invention is controlled by, for example, at least one promoter and at least one terminator, which are defined in more detail below, which are 5 ′ and N, respectively, of the nucleic acid sequence encoding ALD. Present at 3 'position.

Inactivation or reduction of the activity of at least one pdc gene of NADH oxidase inactivates or reduces the ethanol fermentation pathway in yeast. As a result, this induces an unbalanced redox state that is not released by the expression of ALS and ALD. Indeed, the glucose to 2 pyruvate pathway produces 2NADH equivalents, while the conversion of 2 pyruvate to acetoin does not recycle from NADH to NAD ⁺ (see FIG. 1).

  At a specific expression level, the bacterial water-forming NADH oxidase (also referred to as NOXE oxidase or NOXE in this description) enzyme only allows for the equilibration of the redox state, which allows for enhanced stability of this strain. The present inventors have also found that this strain can be enhanced and the acetoin yield is further improved.

Bacterial water-forming NADH oxidase is an enzyme that catalyzes the following reactions:
2NADH + 1 / 2O ₂ → 2NAD ⁺ + H ₂ O

  Preferred water-forming NADH oxidases in the present invention are EC numbers 1.6.3.1 and 1.6.99.3 (NAD (P) H oxidase (also known as H (2) O (2) -forming), double oxidase (dual oxidase), NAD (P) H oxidase, ThOX, THOX2, thyroid NADPH oxidase, thyroid oxidase thyroid oxidase 2 (for EC 1.6.3.1) and NADH dehydrogenase, beta-NADH dehydrogenase dinucleotide, cytochrome C reductase, diaphorase, Dihydrocodehydrogenase I dehydrogenase, dihydronicotinamide adenine dinucleotide dehydrogenase, diphosphopyrinase, DPNH diaphorase, NADH diaphorase, NADH hydrogenase, NADH oxidoreductase, NADH-menadione oxidoreductase, NADH: cytochrome C oxidoreductase, reduction Type diphosphopyridi Nucleotide diaphorase, type 1 dehydrogenase, known by the type I dehydrogenase (about EC1.6.99.3).

  Water-forming NADH oxidases that can be considered in the present invention are described in particular in WO 2006/134277.

  The methods carried out for measuring the activity level of NADH oxidase according to the present invention belong to the common general knowledge of those skilled in the art.

  In this connection, the person skilled in the art may advantageously cite the method described in Lopez DE FELIPE et al. (International Daily Journal, 2001, vol. 11: 37-44 (ISSN 0958-6946)).

  According to a preferred embodiment, the nucleic acid encoding NADH oxidase or NOXE comprises Streptococcus pneumoniae, Lactococcus lactis, Enterococcus faecalis, Lactobacillus brevis and mixtures thereof, and Preferably, it may be a nucleic acid selected from the group comprising S. pneumoniae.

  According to another preferred embodiment, the nucleic acid encoding NADH oxidase or NOXE is from the group consisting of the sequence having at least 78%, preferably at least 80% nucleic acid identity with the nucleic acid sequence SEQ ID NO: 21, 23, 25 and 27. It may be a selected nucleic acid.

  As described herein, a nucleic acid sequence having at least 78% nucleotide identity with a reference nucleic acid sequence is at least 79%, 80%, 81%, 82%, 83%, 84% of said reference nucleic acid sequence. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity Includes nucleic acid sequences.

  According to another particular embodiment, the nucleic acid encoding NADH oxidase is an amino acid selected from the group consisting of sequences having at least 78%, preferably at least 80% identity with SEQ ID NO: 22, 24, 26 and 28. It may be a nucleic acid encoding a sequence.

  As described herein, an amino acid sequence having at least 78% nucleotide identity with a reference amino acid sequence is at least 79%, 80%, 81%, 82%, 83%, 84% with the reference amino acid sequence. , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity Includes amino acid sequence.

  As described above, the expression level of NADH oxidase in the present invention is controlled by, for example, at least one promoter and at least one terminator, as defined in more detail below, which are each 5 ′ of the nucleic acid sequence encoding NADH oxidase. In the 3rd and 3 'positions.

  In addition, the advantageous technical effects described above are linked to the expression level of the NADH oxidase. Indeed, as will become apparent from the following example, not only is the presence of NADH oxidase important, but the level of NADH oxidase expression is also extremely important in acetoin production.

  As mentioned above, the recombinant yeast according to the invention has a reduced pyruvate decarboxylase activity, in which one or more copies of the nucleic acid encoding NADH oxidase or NOXE are inserted in its genome. .

  In this regard, the recombinant yeast according to the invention may in particular contain 1 to 20 copies of nucleic acid encoding NADH oxidase.

  Preferably, the recombinant yeast according to the invention comprises a nucleic acid encoding NADH oxidase of 1 to 12 copies, in particular 2 to 5 copies, preferably 3 to 4 copies, and more preferably equal to 3 copies. Also good.

  According to a particular embodiment, the DNA construct of formula (I) to (III) comprising at least the NOXE gene may be inserted into the endogenous URA3 gene of said recombinant yeast.

  In view of the above, each of the nucleic acids encoding acetolactate synthase, acetolactate decarboxylase, and NADH oxidase is particularly suitable for promoters and terminators so as to avoid unwanted control, such as those defined below. Under control.

For obvious reasons, each of the nucleic acids encoding acetolactate synthase, acetolactate decarboxylase and NADH oxidase is under the control of the same or different promoter.

  The same or different promoters that allow constitutive overexpression of a given gene can be found in the literature (velculescu et al. (1997) Cell 88, 243-251).

Particularly more interesting promoters in the present invention may be selected from the group comprising:
PADH1 (ADH1 gene = SEQ ID NO: 32) from the gene encoding alcohol dehydrogenase,
From the gene encoding glyceraldehyde-3-phosphate dehydrogenase pTDH3 (TDH3 gene = SEQ ID NO: 39),
From the gene encoding the translation elongation factor EF-1 alpha pTEF2.Kl (TEF2 gene = SEQ ID NO: 30),
-PGPM1 from the gene encoding glycerate phosphomutase (GPM1 gene = SEQ ID NO: 33),
From the gene encoding pyruvate decarboxylase pPDC1 (PDC1 gene = SEQ ID NO: 35),
-From the gene encoding Enolase II to pENO2 (ENO2 gene = SEQ ID NO: 29),
・ From the gene encoding the gamma subunit of translation elongation factor eEF1B, pTEF3 (TEF3 gene = SEQ ID NO: 31),
From the gene encoding fructose 1,6-bisphosphate aldolase II pFBA1 (FBA1 gene = SEQ ID NO: 34),
-PPGK1 (PGK1 gene = SEQ ID NO: 36) from a gene encoding 3-phosphoglycerate kinase,
From the gene encoding pyruvate kinase pPYK1 (PYK1 gene = SEQ ID NO: 49),
From the gene encoding triose phosphate isomerase, pTPI1 (TPI1 gene = SEQ ID NO: 50), or from the gene encoding translation elongation factor EF-1 alpha (TEF1 gene = SEQ ID NO: 38).

  In addition, homologous promoters from other closely related yeasts can be derived from other yeasts from the genus Saccharomyces or from other genera such as Candida, Debaryomyces, Pichia or Kluveromyces. It can also be used as a promoter from yeast.

  Synthetic promoters such as those described in Blazeck & Alper (2013) Biotechnol. J. 8 46-58 can also be used.

  More particularly, said promoter, which is the same or different, is preferably characterized by a sequence of nucleic acids selected from the group consisting of sequences having at least 80% nucleic acid identity with the nucleic acid sequences SEQ ID NO: 29 to 39, 49 and 50 May be.

  According to a particular embodiment, each of the nucleic acids encoding acetolactate synthase, acetolactate decarboxylase and NADH oxidase is under the control of the same or different transcription terminator, said transcription terminator being a nucleic acid of SEQ ID NO: 40 to 48 Characterized by a sequence of nucleic acids selected from the group consisting of sequences having at least 80% nucleic acid identity with the sequence.

Terminator For obvious reasons, each of the nucleic acids encoding acetolactate synthase, acetolactate decarboxylase and NADH oxidase is linked to the same or different transcription terminator (which may also be referred to herein as “terminator”).

  The same or different transcription terminators can be found in the document Yamanishi et al. (2013) ACS synthetic biology 2, 337-347.

Terminators that are particularly more interesting in the present invention may be selected from the group comprising:
From the gene encoding triose phosphate isomerase tTPI1 (TPI1 gene = SEQ ID NO: 44),
From the gene encoding O-acetylhomoserine-O-acetylserine sulfhydrylase tMET25 (Met25 gene = SEQ ID NO: 45),
TADH1 from the gene encoding alcohol dehydrogenase (ADH1 gene = SEQ ID NO: 43),
TENO2 from the gene encoding Enolase II (ENO2 gene = SEQ ID NO: 46),
From the gene encoding glyceraldehyde-3-phosphate dehydrogenase, isozyme 2, tTDH2 (TDH2 gene = SEQ ID NO: 40),
・ From a gene encoding 3-phosphoglycerate kinase, tPGK1 (PGK1 gene = SEQ ID NO: 48),
TCYC1 (= SEQ ID NO: 41),
TMET3 (= SEQ ID NO: 47), tTDH3 (= SEQ ID NO: 42), and tDIT1 (= SEQ ID NO: 43).

  More particularly, said terminator, which is the same or different, may be characterized by a sequence of nucleic acids preferably selected from the group consisting of sequences having at least 80% identity with SEQ ID NO: 32 to 40 and 43 .

Recombinant yeast In general, yeast can grow rapidly, can be cultured at a higher density compared to bacteria, and does not require an aseptic environment in an industrial setting. Furthermore, yeast cells are a much simplified process for product extraction and purification and can be more easily separated from the culture medium compared to bacterial cells.

  Preferentially, the yeast of the present invention comprises Saccharomyces, Candida, Ashbya, Dekkera, Pichia (Hansenula), Debariomyces, Clavispora. Clavispora) It may be selected from the genus (Cryptococcus) or Malassezia.

  More preferentially, the yeast may be a Crabtree positive yeast of the genus Saccharomyces, Deckella, Schizosaccharomyces, Kleiberomyces, Torlaspora genus Digosaccharomyces, or Brettanomyces.

  More preferentially, the yeast is a budding yeast, Saccharomyces boulardii, Saccharomyces douglasii, Saccharomyces bayanus, or Zigosaccharzomyspoi ), Dekkera brucelensis, Dekkera intermedia, Brettanomycces custersii, Brettanomycces intermedius, Kluyveromyces themotolerens), Torulaspora globosa, Torulaspora glabrata species.

  More preferentially, the recombinant yeast may belong to the genus Saccharomyces, and preferably to the budding yeast species.

  As mentioned above, the recombinant yeast according to the invention comprises at least one nucleic acid encoding at least one DNA construct selected from the group comprising formulas (I) to (III), and preferably encoding ALS and / or ALD. Having pyruvate decarboxylase activity which is reduced by insertion of at least one said DNA construct comprising only

  According to a preferred embodiment, the recombinant yeast may be a recombinant budding yeast and pyruvate decarboxylase activity is reduced by disruption of only two pdc genes. More preferably, the disrupted pdc gene may be selected from the group consisting of pdc1, pdc5, pdc6 and mixtures thereof, and preferably pdc1 and pdc6.

  The methods practiced to insert the gene and more particularly the specific DNA construct with the pyruvate decarboxylase gene are within the common general knowledge of those skilled in the art. Related methods are described in more detail by the following examples.

Most preferred embodiment Advantageously, the nucleic acid encoding the enzyme practiced in the present invention is ALS.Bs, ALS.Pp, ALD.Ll, ALD.Ea, NOXE.spn, NOXE.Ll and mixtures thereof. Is advantageously selected.

According to a preferred embodiment, the recombinant yeast according to the invention is characterized in that the endogenous pyruvate decarboxylase activity is reduced by disruption of at least two pdc genes, in particular by disruption of the pdc1 and pdc6 genes. It may be characterized by belonging to a yeast species, where:
-One pdc gene, preferably the pdc1 gene, has the following formula (IIe):
_{5 '- [(prom5) y1} -ALS.Bs-term5] x5 - [prom1-ALS.Bs-term1] x1 - [prom2-ALD.Ll- (term2) z1] x2 -3' (IIe)
Destroyed by insertion of the DNA construct of
-Apart from the disrupted pdc gene mentioned above, at least the other pdc gene, and preferably the pdc6 gene, has the following formula (IIf '):
_{5 '- [(prom5) y1} -ALS.Pp-term5] x5 - [prom1-ALS.Pp-term1] x1 - [prom2-ALD.Ea- (term2) z1] x2 -3' (IIf ')
Destroyed by insertion of the DNA construct of
As well as the following formula (IIf ''):
5 '-[(prom3) _y2 -NOXE.Ll- (term3) _z2 ] _x3 -3' (IIf '')
The DNA construct of is inserted into the URA3 gene,
Where:
-prom1, prom2, prom3, prom5, term1, term2, term3, term5, `` y1 '', `` y2 '', `` z1 '' and `` z2 '' are defined above, for example, ALS.Bs, ALS.Pp, ALD .Ll, ALD.Ea and NOXE.Ll are defined, for example, in Table 1 below.
-Each of `` x1 '', `` x2 '' and `` x3 '' independently of one another is in the range 0 to 50, preferably 0 to 20, preferably 0 to 10, more particularly 0 to 3, and in particular Represents an integer equal to 1;
-"X3" represents an integer from 0 to 50, preferably 0 to 20, preferably 0 to 12, more particularly 2 to 5, preferably 3 to 4 and more preferably equal to 3.
However, the recombinant yeast contains at least one nucleic acid encoding each ALS, ALD and NOXE, provided that each DNA construct of formulas (IIe) and (IIf ′) encodes ALS and ALD in more detail. Each comprising at least one nucleic acid.

In terms of the above, although implicitly disclosed, between each formula (IIe) and (IIf ′):
-“X1” to “x3”, “x5”, “y1”, “y2”, “z1” and “z2”; and / or
-It is specified that the promoter and / or terminator for each copy of nucleic acid for the gene under consideration may be the same or different.

According to certain preferred embodiments, the recombinant yeast according to the present invention comprises a genus Saccharomyces, wherein the endogenous pyruvate decarboxylase activity is reduced by disruption of at least two pdc genes, in particular by disruption of the pdc1 and pdc6 genes, It may be characterized in particular by belonging to the budding yeast species, where:
-One pdc gene, preferably the pdc1 gene has the following formula (IIg):
5 '-[ALS.Bs-tTDH2] _1- [pENO2-ALD.Ll-tCYC1] ₁ -3' (IIg)
Destroyed by insertion of DNA constructs
-Apart from the disrupted pdc gene mentioned above, at least other pdc genes, and preferably the pdc6 gene, have the following formula (IIh '):
5 '-[pADH1-ALS.Pp-tDPI1] _1- [pTDH3-ALD.Ea-tMET25] ₁ -3' a (IIh ')
Destroyed by insertion of DNA constructs
As well as a DNA construct of the following formula (IIh ''):
5 '-[pENO2-NOXE. Ll-tPGK1] ₁₂ -3' (IIh '')
Is inserted into the URA3 gene,
here:
The `` ALS.Bs '' gene of the DNA construct of formula (IIg) is under the control of the promoter of the pdc gene into which the DNA construct of formula (IIg) is inserted;
-pENO2, pADH1, pTDH3, tTDH2, tCYC1, tDPI1, tMET25 and tPGK1, for example, are defined in this description and in more detail in the sequence listing below,
-ALS.Bs, ALS.Pp, ALD.Ll, ALD.Ea and NOXE.Ll are defined, for example, in the following table 1 (Table 1) and in more detail in the following sequence listing.

  According to certain embodiments, the recombinant yeast according to the present invention may be further modified to optimize acetoin production.

Use of alternative sources of sugar:
The direct use of alternative sources of sugar, such as starch, further requires overexpression of exogenous α-amylase and glucoamylase in yeast (buscke et al., Biosource technology 2013).

Sugar uptake and transport-Improving C5 sugar uptake and transport The uptake and transport of pentose by recombinant microorganisms is an important issue for industrial processes because the C5 sugar is hydrolyzed lignocellulosic biomass. It is because it is a main component of the. Natural strains of budding yeast, such as many other types of yeast, cannot utilize either xylose or arabinose as fermentation substrates (Hahn-Hagerdal et al., 2007; Jin et al., 2004). Interestingly, the strain can take up the sugar even though xylose is not a natural substrate (Hamacher et al., 2002).

  Budding yeasts GAL2, HXT1, HXT2, HXT4, HXT5, and HXT7 catalyze xylose uptake because they have broad substrate specificity (Hamacher et al., 2002; Saloheimo et al., 2007; Sedlak & Ho 2004). However, their affinity for xylose is much lower than that for glucose, and xylose uptake by the transporter is strongly inhibited by glucose (Saloheimo et al., 2007).

  Some changes are necessary to obtain strains that can grow and consume xylose and / or arabinose. These different modifications are part of the present invention.

Overexpression of heterologous xylose transporters To improve xylose and arabinose uptake, recombinant acetoin producing strains must be modified to express heterologous genes encoding xylose or arabinose transporters. For example, the genes GXF1, SUT1 and AT5g59250 from Candida intermedia, Pichia stipitis and Arabidopsis thaliana, respectively, are overexpressed to improve xylose utilization by yeast (Runquist Et al., 2010).

Overexpression of pathways involved in xylose and arabinose metabolism Yeast strains can take up their sugars even though xylose is not a natural substrate. Even though genes for xylose assimilation are present in budding yeast, they are not expressed at sufficient levels to allow significant sugar assimilation. Therefore, genetic modification is necessary to improve the anabolism of pentose sugars. There is a need for all enzymes that allow the conversion of xylose or arabinose to xylitol as well as enzymes that convert xylitol to xylulose and xylulose to xylulose-5-phosphate. Either homologous genes from the xylose and arabinose pathways must be overexpressed or heterologous genes from bacteria must be overexpressed.

In one embodiment of the present invention, xylose uptake by the strain and its anabolism is improved, for example, by overexpressing:
1) P. stippitis associated with overexpression of XYL2 encoding xylitol dehydrogenase from P. stippitis and combined with overexpression of genes XKS1 or XYL3 encoding xylulokinase from budding yeast and P. stippitis, respectively. And a gene XYL1 or GRE3 encoding a budding yeast aldolase reductase,
2) The gene xylA encoding xylose isomerase from bacteria or Piromyces associated with overexpression of the gene XKS1 or XYL3 encoding xylulokinase from Saccharomyces cerevisiae and P. stippitis, respectively.

In another embodiment of the invention, arabinose uptake by the strain and its assimilation are improved, for example, by overexpressing:
1) Overexpression of XYL2 encoding xylitol dehydrogenase from P. stipitis, associated with ladl encoding L-arabinitol 4-hydrogenase from Trichoderma reesei and Ixrl encoding L-xylulose reductase, And, in addition, homologous genes XYL1 or GRE3 encoding P. stippitis and Saccharomyces cerevisiae aldolase reductase, respectively, combined with overexpression of genes XKS1 or XYL3 encoding xylulokinase from Saccharomyces cerevisiae and P. stipits, respectively.
2) Heterologous genes araA and araB encoding bacterial arabinose isomerase and ribulose kinase.

Optimization of the pentose phosphate pathway This can be done by overexpression of at least one gene belonging to the non-oxidative pentose phosphate pathway from yeast strains; TALI, TKL1, RKL1 and RPE1.

Optimization of availability of NAPDH cofactors required for enzymes involved in C5 sugar metabolism This is accomplished by expressing E. coli transhydrogenase in yeast strains. The genes udhA and / or pntAB from E. coli are overexpressed in the production strain.

Prevention of glucose consumption for glycerol synthesis:
This can be done by disruption of the GPD1 gene encoding glycerol-3-phosphate dehydrogenase EC 1.1.1.8. (GPDH).

  The present invention according to this embodiment is particularly interesting in terms of yield in acetoin, despite the fact that disruption of the GPD1 gene leads to the removal of enzymatic activity that consumes NADH in preference to NAD. In order to balance the redox disequilibrium thus generated, GPD1-disrupted strains may require additional expression of NOXE.

  According to a particular embodiment, the recombinant strain according to the invention reduces glucose suppression as disclosed in WO 2011/041426 or Kim et al. (Bioresource Technology, vol. 146, 2013: 274). Does not include any genetic modifications that have an effect.

  According to a particular embodiment, the recombinant strain according to the invention can be any genetic strain that allows expression of any xylose anabolic pathway, as disclosed in Kim et al. (Journal of Biotechnology, 2014). Does not include modifications.

Culture conditions The invention also relates to the use of recombinant yeast, for example as defined above, for the production of acetoin and / or derivatives thereof, in particular methyl vinyl ketone (MVK).

The present invention further relates to a method for the production of acetoin comprising the following steps:
-Preparing a recombinant microorganism as described above, culturing the recombinant microorganism in a culture medium containing a carbon source, and
-The process of recovering acetoin.

  Typically, the microorganisms of the invention may be grown in a suitable culture medium at a temperature in the range of about 20 ° C to about 37 ° C, preferably in the range of 27 to 34 ° C.

  When the recombinant yeast according to the invention belongs to the budding yeast species, the temperature can advantageously range from 27 to 34 ° C. in a suitable culture medium.

  Suitable growth media for yeast include yeast nitrogen base, ammonium sulfate, and dextrose (as a carbon / energy source) or YPD medium, a blend of peptone, yeast extract, and dextrose in the optimal proportions for most growth. , Common commercial conditioned media such as broth. Other defined or synthetic growth media may be used and suitable media for the growth of specific microorganisms are known to those skilled in the microbiology or fermentation science arts.

  The term “appropriate culture medium” is defined above.

  Examples of known culture media for recombinant yeast according to the present invention are known to those skilled in the art and are presented in the literature D. Burke et al., Methods in yeast Genetics-A cold spring harbor laboratory course Manual (2000). Yes.

  A suitable pH range for the fermentation may be between pH 3.0 and pH 7.5, where pH 4.5 to pH 6.5 are preferred as initial conditions.

  Fermentation may be performed under aerobic conditions or microaerobic conditions.

  The amount of product in the fermentation medium can be determined using several methods known in the art such as, for example, high performance liquid chromatography (HPLC) or gas chromatography (GC).

  The process may utilize a fermentation batch process. Classic batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation, the medium is inoculated with the desired organism, allowing the fermentation to occur without adding anything to the system. Typically, however, a “batch” fermentation is a batch with respect to the addition of a carbon source, and many attempts are made under control factors such as temperature, pH and oxygen concentration. In a batch system, the metabolite and biomass composition of the system always changes until the time when the fermentation stops. Within batch culture, cells progress from a static induction phase to a high growth log phase and finally a stationary phase where the growth rate is reduced or stopped. When untreated, cells in the stationary phase eventually die. Cells in the log phase are generally responsible for most of the production of the final product or intermediate.

A fed batch system may also be used in the present invention. A fed batch system is similar to a typical batch system, with the exception that the carbon source substrate is added in small increments as the fermentation proceeds. The fed-batch system is useful when catabolite repression (eg, glucose suppression) tends to inhibit cellular metabolism and when it is desirable to have a limited amount of substrate in the medium. Measuring the actual substrate concentration in a fed-batch system is difficult and is therefore estimated on the basis of changes in measurable factors such as pH, dissolved oxygen and partial pressure of waste gases such as CO ₂ .

  Fermentation is common and well known in the art and can be found in Sunderland et al. (1992), examples of which are incorporated herein by reference. Although the present invention is performed in a batch mode, it is contemplated that the method is adaptable to continuous fermentation.

  Continuous fermentation is an open system in which a defined fermentation medium is continuously added to the bioreactor and an equal amount of conditioned medium is removed simultaneously with processing. Continuous fermentation generally maintains the culture at a constant high density where the cells are primarily in log phase growth.

  Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method maintains limited nutrients, such as carbon source or nitrogen levels, at a fixed rate and allows all other parameters to vary. In other systems, several factors that affect growth can be changed continuously while keeping the cell concentration measured by medium turbidity constant. Because continuous systems strive to maintain steady state growth, cell defects due to the flowed out media must be balanced against the rate of cell growth during fermentation. Methods for regulating nutrients and growth factors for continuous fermentation processes and techniques for maximizing the rate of product formation are well known in the field of industrial microbiology.

  It is contemplated that the present invention can be practiced using batch, fed-batch or continuous processes and that any known mode of fermentation is suitable. It is further contemplated that the cells can be immobilized on a substrate as a whole cell catalyst and subjected to fermentation conditions for production.

  In order to still improve acetoin production, a specific embodiment is the step of culturing the recombinant yeast cells in a suitable culture medium as described above, wherein the culture medium comprises an optimal amount of carbon source, in particular You may consist of a process containing glucose.

  Preferably, the cells are cultured in an optimal culture medium for only a portion of the total culture duration. In some embodiments, the yeast cells are incubated in the optimal culture medium for 10 hours or more after the start of culture, including 11, 12, 13, 14, 15 or 16 hours or more after the start of culture. .

  Preferably, the cells are cultured in such an optimal culture medium for a period ranging from 5 to 15 hours, including 6 to 10 hours, such as 8 hours, after the start of the culture.

  In a preferred embodiment, a carbon source is included in the optimal culture medium composed of glucose. In a preferred embodiment, the optimal culture medium comprises 12% w / w or more glucose, comprising 15% w / w or more glucose. In a preferred embodiment, the optimal culture medium comprises at most 35% w / w glucose, at most 40% w / w glucose.

  Therefore, in the above preferred embodiment, the method for producing acetoin according to the present invention comprises an intermediate step comprising culturing yeast cells in the optimal culture medium between steps (a) and (c). (b) may further be included.

Purification of acetoin According to a particular embodiment of the invention, the fermentative production of acetoin comprises a step of isolation of acetoin from the culture medium. The process of recovering acetoin from the culture medium is a routine task for those skilled in the art. It may be achieved by several techniques well known in the art including, but not limited to, distillation, gas stripping, pervaporation or liquid extraction. Experts in the field know how to adapt the parameters of each technique depending on the characteristics of the material to be separated.

  Yeast as a microorganism model in the present invention is retained because the synthesized acetoin is completely excreted and transported out of the cell, thus simplifying the purification process.

  The synthesized acetoin may be collected by distillation. Distillation may be accompanied by optional components that differ from the culture medium to facilitate the isolation of acetoin by forming an azeotrope with water in particular. This optional component is an organic solvent such as cyclohexane, pentane, butanol, benzene, toluene, trichloroethylene, octane, diethyl ether or mixtures thereof.

  Gas stripping is accomplished by gas stripping selected from helium, argon, carbon dioxide, hydrogen, nitrogen or mixtures thereof.

  Liquid extraction is accomplished with an organic solvent as the hydrophobic phase, such as pentane, hexane, heptane, dodecane.

  Starting from acetoin produced by the recombinant yeast cells described herein, acetoin derivatives, including methyl vinyl ketone, can be obtained through various methods well known by those skilled in the art.

  Throughout the description, including the claims, the expression “comprising” should be understood to be synonymous with “comprising at least one” unless otherwise specified. It is.

  In addition, the expression `` formulas (I) to (III) '' depending on the context to be considered means the DNA construct of formulas (I), (II) and (III), unless indicated otherwise, It also means (IIa), (IIb), (IIc), (IId), (IIe), (IIf ′), (IIf ″), (IIg), (IIh ′) and / or (IIh ″).

  The terms "between ... and ..." and "ranging from ... to ..." Unless specified, it should be understood to encompass limit values.

  The following examples and figures are provided by way of illustration and do not imply a limitation on the invention.

a) Protocol for producing a recombinant budding yeast strain according to the present invention All the following recombinant budding yeast strains to be implemented are from standard strain W303 (Thomas and Rothstein (1989), Cell. 56, 619-630). It was constructed using standard yeast molecular genetics procedures (Methods in yeast Genetics-A cold spring harbor laboratory course Manual (2000) by D. Burke, D. Dawson, T. Stearns CSHL Press).

  In these strains, pyruvate decarboxylase activity may be reduced by disruption of at least one pdc gene (pdc1, pdc5, pdc6) or by replacement of their cognate transcription promoter by a weak promoter.

  In the most efficient strain, only pdc1 and pdc6 were deleted.

  Various exogenous enzymes were expressed in the considered recombinant budding yeast strains. They were chosen according to their Michaelis Menten enzyme parameters when available (see table 1 below). High kcat for high efficiency and various Km covering different concentrations in the substrate. Paenibacillus polymixer was chosen because this microorganism is a natural 2,3-butanediol-producing bacterium and is a product derived directly from acetoin. Thus, it has an enzyme that efficiently catalyzes acetoin synthesis.

  The gene nomenclature associated with the exogenous enzymes acetolactate synthase, acetolactate decarboxylase and water-forming NADH oxidase performed is shown in Table 1 below.

  These genes are designated by an enzyme acronym followed by an organism acronym of origin:

In addition, the following genotypes for better understanding:
-ade2, his3, leu2, trp1 and ura3 are auxotrophic marker genes.
-Lower case letters mean that the genes considered are inactive, upper case letters reflect active genes.
-"::": following the gene name means that the gene is interrupted by what follows (if more than one gene is inserted, they are noted in square brackets []) . Gene disruption is accompanied by an overall deletion of the coding sequence, but the promoter is conserved. As a result, genes followed by “::” are inactive and are noted in lower case. If not specified, transcription of the inserted gene is controlled by the promoter of the disrupted gene.
-“Gene.Kl” means that the gene is derived from Kleiberomyces lactis.
-Transcriptional promoters that allow constitutive overexpression of a given gene are found in the literature (velculescu et al. (1997) Cell 88, 243-251). Promoters as used herein are designated by their cognate gene name followed by “p”. Their respective SEQ ID NOs are also described below.
-A transcription terminator is also placed after each gene. In order to avoid unnecessary regulatory promoter and terminator frames, some inserted genes were not obtained from the same original gene. Terminators as used herein are designated by their cognate gene name followed by “t”. Their respective SEQ ID NOs are also described below.

  The cluster of genes described above was incorporated into recombinant yeast at once using the ability of yeast to efficiently recombine free DNA ends with sequence homology.

  Recombinant yeast was obtained according to published methods available to those skilled in the art. In particular, Shao et al. (Nucleic Acids Research, 2009, Vol. 37, No. 2: e16) and Shao et al. (Methods in Enzymology, 2012 Elsevier Inc., Vol. : 203).

  More particularly, the coding sequence to be cloned was artificially synthesized. For heterologous sequences (non-yeast), the nuclear sequence was modified to obtain synonymous coding sequences using yeast codon usage. Each synthetic sequence was cloned between a transcriptional promoter and transcriptional terminator using restriction enzymes and classical cloning techniques. Each promoter sequence is preceded by a 50-200 nucleotide sequence homologous to the upstream gene terminator sequence. Similarly, the terminator of each gene (promoter-coding sequence-gene containing terminator) is immediately followed by a sequence homologous to the immediately following gene. Thus, each of the units incorporated has a 50-200 nucleotide overlap with both the unit upstream and unit downstream. For the first unit, the promoter is preceded by 50-200 nucleotides homologous to the yeast chromosomal nucleotide for the locus to be integrated. Similarly, for the last unit, the terminator is followed by 50-200 nucleotides homologous to the yeast chromosomal nucleotide for the locus to be integrated.

  Each unit is then PCR amplified from the plasmid construct to yield a linear DNA X unit with overlapping sequences. One of these genes is an auxotrophic marker for selecting recombination events. All linear fragments are transformed into yeast at once and the recombinant yeast is selected for auxotrophy associated with the marker used. The integrity of the sequence is then verified by PCR and sequencing.

b) Regarding ALS and ALD enzymes
ALS and ALD enzymes were not evaluated individually, but were evaluated in pairs (ALS + ALD) through the yield of acetoin. Three exogenous ALD and ALS were chosen according to their kinetic parameters and were designated ALS.Nt, ALS.Pp, ALS.Bs and ALD.Bb, ALD.Ll, ALD.Ea (see above) ).

  Eight of the nine possible combinations of ALS and ALD were inserted jointly into the chromosome of the ura3-yeast strain behind the promoter and followed by one terminator.

Insertion of these two genes disrupts the pdc1 gene. The URA3 marker gene is inserted concomitantly to select transformants. The ALS / ALD combination was inserted into strain YA747, a W303 derivative with the following genotype:
YA747: MAT-a, ade2, bdh1 :: TRP1.Kl, his3, leu2, pdc1 :: HIS5.Sp, pdc6 :: LEU2.Kl, trp1, ura3.

The following strains were constructed:
YA768: MAT-a, ade2, bdh1 :: TRP1.Kl, his3, leu2, pdc1 :: [-ALS.Bs-tTPI1, pTDH3-ALD.Ea-tMET25, URA3.Kl], pdc6 :: LEU2.Kl, trp1, ura3
NB: In this case, the gene “ALS.Bs” is under the control of the natural promoter of pdc1, ie the promoter pPDC1.
YA769: MAT-a, ade2, bdh1 :: TRP1.Kl, his3, leu2, pdc1 :: [-ALS.Nt-tTPI1, pTDH3-ALD.Ea-tMET25, URA3.Kl], pdc6 :: LEU2.Kl, trp1, ura3
YA770: MAT-a, ade2, bdh1 :: TRP1.Kl, his3, leu2, pdc1 :: [-ALS.Pp-tTPI1, pTDH3-ALD.Ea-tMET25, URA3.Kl], pdc6 :: LEU2.Kl, trp1, ura3
YA771: MAT-a, ade2, bdh1 :: TRP1.Kl, his3, leu2, pdc1 :: [-ALS.Nt-tTPI1, pTDH3-ALD.Bb-tMET25, URA3.Kl], pdc6 :: LEU2.Kl, trp1, ura3
YA772: MAT-a, ade2, bdh1 :: TRP1.Kl, his3, leu2, pdc1 :: [-ALS.Nt-tTPI1, pTDH3-ALD.Ll-tMET25, URA3.Kl], pdc6 :: LEU2.Kl, trp1, ura3
YA773: MAT-a, ade2, bdh1 :: TRP1.Kl, his3, leu2, pdc1 :: [-ALS.Pp-tTPI1, pTDH3-ALD.Ll-tMET25, URA3.Kl], pdc6 :: LEU2.Kl, trp1, ura3
YA810: MAT-a, ade2, bdh1 :: TRP1.Kl, his3, leu2, pdc1 :: [-ALS.Bs-tTPI1, pTDH3-ALD.Bb-tMET25, URA3.Kl], pdc6 :: LEU2.Kl, trp1, ura3
YA811: MAT-a, ade2, bdh1 :: TRP1.Kl, his3, leu2, pdc1 :: [-ALS.Pp-tTPI1, pTDH3-ALD.Bb-tMET25, URA3.Kl], pdc6 :: LEU2.Kl, trp1, ura3

  All these strains were grown in 8% glucose YPA (1% yeast extract, 2% Bacto peptone, 0.1 mM adenine, 8% glucose) for 24 hours. They were harvested and the amounts of acetoin and ethanol were determined according to standard methods specifically adapted from items in Gonzales et al. (2010) Applied and environmental Microbiology 76 670-679.

  For some strains, several clones were assayed, the last number after the “-” is the clone number. It is noted that the endogenous bdh enzyme is destroyed and 2,3-BDO is not produced.

  Ethanol and acetoin production is monitored according to standard methods and Gonzales et al. (2010) Applied and environmental Microbiology 76 670-679.

Results Table 2 below shows the acetoin production of the strains tested above.

  From these results, it can be concluded that the best enzymes that enhance acetoin production are ALS Pp, ALS Nt, ALD Ea and ALD Bb, which seem to be the most efficient enzymes in practice. Can be.

c) Advantageous technical effect of NOXE enzyme on acetoin yield
Recombinant yeast according to YA1609 has been prepared.
YA1609: MAT-a, his3, leu2, pdc1 :: [-ALS.Bs-tTDH2- pENO2-ALD.Ll-tCYC1, HIS5.Sp], pdc6 :: [pADH1-ALS.Pp-tDPI1, pTDH3-ALD. Ea-tMET25, LEU2.Kl], trp1, ura3 :: [pENO2-NOXE.Ll-tPGK1, URA3] x12

  YA1609-1, YA1609-2 and YA1609-4 are different clones from this strain YA1609.

  It is noted that these recombinant yeasts always have their natural endogenous BHH activity.

  These strains were then assayed for acetoin production in 16% glucose YPA (1% yeast extract, 2% Bacto peptone, 0.1 mM adenine, 8% glucose) under the same conditions as above.

  Ethanol and acetoin production is monitored according to standard methods and Gonzales et al. (2010) Applied and environmental Microbiology 76 670-679.

The results are reported in Table 3 below.

  These results indicate that the presence of NOXE leads to a very meaningful accumulation of acetoin.

YA1609-4 is now performed in the same culture medium as described above. However, the assay depends on the following parameters:
Environment: 0.5l YPA, 16% glucose.
Sixteen hours after the start of cell culture, the cells were incubated for a period of 8 hours in culture medium containing a final concentration of 16% w / w glucose.
Stirring: 800rpm (2 blades), 1.9ms ^-1
Temperature: 30 ℃
Air: 0.15L / min (0.3vvm)

  Cells were then assayed for acetoin production. Ethanol and acetoin production is monitored according to standard methods and Gonzales et al. (2010) Applied and environmental Microbiology 76 670-679.

Results The results are reported in Table 4 below.

  These results indicate that the culture parameters considered can also affect acetoin production.

d) Further examples of recombinant yeast producing acetoin Further recombinant yeasts have been prepared and are described below.
YA1573-4: MAT-a, his3, leu2, pdc1 :: [-ALS.Bs-tTDH2, pENO2-ALD.Ll-tCYC1, HIS5.Sp], pdc6 :: [pADH1-ALS.Pp-tDPI1, pTDH3- ALD.Ea-tMET25, pTEF2-TRP1.Sc-tADH1, pGMP1-BDH.Sc-tENO2], trp1, ura3 :: [pENO2-NOXE.Ll-tPGK1, URA3] x12
YA1609-4: MAT-a, his3, leu2, pdc1 :: [-ALS.Bs-tTDH2, pENO2-ALD.Ll-tCYC1, HIS5.Sp], pdc6 :: [pADH1-ALS.Pp-tTPI1, pTDH3- ALD.Ea-tMET25, LEU2.Kl], trp1, ura3 :: [pENO2-NOXE.Ll-tPGK1, URA3] x12
YA1661-2: MAT-a, his3, leu2, pdc1 :: [-ALS.Bs-tTDH2, pENO2-ALD.Ll-tCYC1, HIS5.Sp], pdc6 :: [pADH1-ALS.Pp-tTPI1, pTDH3- ALD.Ea-tMET25, pENO2-NOXE.Spn-tPGK1], trp1, ura3 :: [pENO2-NOXE.Ll-tPGK1, URA3] x12.

  It is noted that these recombinant yeasts always have their natural endogenous BHH activity.

  These strains were then assayed for acetoin production in 16% glucose YPA (1% yeast extract, 2% Bacto peptone, 0.1 mM adenine, 8% glucose) under the same conditions as above.

  Ethanol and acetoin production is monitored according to standard methods and Gonzales et al. (2010) Applied and environmental Microbiology 76 670-679.

Results The results are reported in Table 5 below.

  These results in Table 5 show that the presence of multiple nucleic acids encoding NOXE in recombinant yeast resulted in an increase in acetoin accumulation compared to recombinant yeast transformed with a single nucleic acid encoding NOXE. Indicates that an increase is led.

e) Prevention of acetoin escape toward 2,3-BDO At least one of the following endogenous bdh1, bdh2, adh1, adh3 and / or adh4 genes may be inactivated. In some strains, auxotrophic markers were reintroduced to make them prototrophs. They all have the same promoter and terminator compared to YA1660:
YA1660-2, YA1660-3, YA1660-4, and YA1711-16C: MAT-a, his3, leu2, pdc1 :: [-ALS.Bs-tTDH2, pENO2-ALD.Ll-tCYC1, HIS5.Sp], pdc6 :: [pADH1-ALS.Pp-tTPI1, pTDH3-ALD.Ea-tMET25, pENO2-NOXE.Spn-tPGK1, LEU2.Kl], trp1, ura3 :: [pENO2-NOXE.Ll-tPGK1, URA3] x12.
YA1711-45c: Mat-α, his3, leu2, pdc1 :: [-ALS.Bs-tTDH2, pENO2-ALD.Ll-tCYC1, HIS5.Sp], pdc6 :: [pADH1-ALS.Pp-tTPI1, pTDH3- ALD.Ea-tMET25, pENO2-NOXE.Spn-tPGK1, LEU2.Kl], trp1, ura3 :: [pENO2-NOXE.Ll-tPGK1, URA3] x12
YA1711-13A, YA1711-16B: Mat-a, bdh1 :: LEU2.Kl, bdh2 :: HIS5.Sp, his3, leu2, pdc1 :: [ALS.Bs-ALD.Ll-HIS5.Sp], pdc6 :: [ALS.Pp-ALD.Ea-NOXE.Spn-LEU2.Kl], trp1, ura3 :: [NOXE.Ll-URA3] x12
YA1711-19A and YA1711-46A: Mat-α, bdh1 :: LEU2.Kl, bdh2 :: HIS5.Sp, his3, leu2, pdc1 :: [ALS.Bs-ALD.Ll-HIS5.Sp], pdc6 :: [ALS.Pp-ALD.Ea-NOXE.Spn-LEU2.Kl], trp1, ura3 :: [NOXE.Ll-URA3] x12
YA1822-1 and YA1822-2: Mat-α, bdh1 :: LEU2.Kl, bdh2 :: HIS5.Sp, his3, leu2, pdc1 :: [ALS.Bs-ALD.Ll-HIS5.Sp], pdc6 :: [ALS.Pp-ALD.Ea-NOXE.Spn-LEU2.Kl], trp1 :: TRP1, ura3 :: [NOXE.Ll-URA3] x12
YA 1870-10A: Mat-a, adh3 :: TRP1.Kl, bdh1 :: LEU2.Kl, bdh2 :: HIS5.Sp, his3, leu2, pdc1 :: [ALS.Bs-ALD.Ll-HIS5.Sp] , Pdc6 :: [ALS.Pp-ALD.Ea-NOXE.Spn-LEU2.Kl], trp1, ura3 :: [NOXE.Ll-URA3] x12
YA 1870-11B: Mat-α, adh3 :: TRP1.Kl, bdh1 :: LEU2.Kl, bdh2 :: HIS5.Sp, his3, leu2, pdc1 :: [ALS.Bs-ALD.Ll-HIS5.Sp] , Pdc6 :: [ALS.Pp-ALD.Ea-NOXE.Spn-LEU2.Kl], trp1, ura3 :: [NOXE.Ll-URA3] x12
YA1870-10B: Mat-α, adh1 :: TRP1.Kl, bdh1 :: LEU2.Kl, bdh2 :: HIS5.Sp, his3, leu2, pdc1 :: [ALS.Bs-ALD.Ll-HIS5.Sp], pdc6 :: [ALS.Pp-ALD.Ea-NOXE.Spn-LEU2.Kl], trp1, ura3 :: [NOXE.Ll-URA3] x12
YA1870-11A: Mat-α, adh1 :: TRP1.Kl, bdh1 :: LEU2.Kl, bdh2 :: HIS5.Sp, his3, leu2, pdc1 :: [ALS.Bs-ALD.Ll-HIS5.Sp], pdc6 :: [ALS.Pp-ALD.Ea-NOXE.Spn-LEU2.Kl], trp1, ura3 :: [NOXE.Ll-URA3] x12
YA1870-2A and YA1870-3D: Mat-a, adh1 :: TRP1.Kl, adh3 :: TRP1.Kl, bdh1 :: LEU2.Kl, bdh2 :: HIS5.Sp, his3, leu2, pdc1 :: [ALS. Bs-ALD.Ll-HIS5.Sp-loxP], pdc6 :: [ALS.Pp-ALD.Ea-NOXE.Spn-LEU2.Kl-loxP], trp1, ura3 :: [NOXE.Ll-URA3] x12

  All the above strains and YA1609-4 and YA1661-2 and diploid combinations of these strains should be considered as strains with improved acetoin yield.

f) Further example showing prevention of acetoin escape towards 2,3-BDO Strain YA1711-19A was grown in 2 liters of yeast extract 2%, sucrose 10% in a 3 l fermentor for 0 to 24 hours, Then 600 g glucose was added slowly (20 h at 30 g / l).

  The following strains, all consisting of strains into which exogenous ALS, ALD and NOXE were introduced, were incubated in 500 ml Erlenmeyer flasks, 24 hours and 48 hours, 30 ° C., yeast extract 2% and sucrose 30 Shake in%:

Sequence listing

  Sequence number 1 (= ADN ALS.Bs)

  SEQ ID NO: 2 (= amino acid ALS.Bs)

  Sequence number 3 (= ADN ALS.Nt)

  SEQ ID NO: 4 (= amino acid ALS.Nt)

  Sequence number 5 (= ADN ALS.Pp)

  SEQ ID NO: 6 (= amino acid ALS.Pp)

  Sequence number 7 (= ADN ALD.Bb)

  SEQ ID NO: 8 (= amino acid ALD.Bb)

  Sequence number 9 (= ADN ALD.Ea)

  SEQ ID NO: 10 (= amino acid ALD.Ea)

  Sequence number 11 (= ADN ALD.Ll)

  SEQ ID NO: 12 (= amino acid ALD.Ll)

  Sequence number 13 (= ADN NOXE.Ll)

  SEQ ID NO: 14 (= amino acid NOXE.Ll)

  Sequence number 15 (= ADN NOXE.spn)

  SEQ ID NO: 16 (= amino acid NOXE.spn)

  Sequence number 17 (= ADN NOXE.Ef)

  SEQ ID NO: 18 (= amino acid NOXE.Ef)

  Sequence number 19 (= ADN NOXE.Lb)

  SEQ ID NO: 20 (= amino acid NOXE.Lb)

  SEQ ID NO: 21 (= pENO2)

  SEQ ID NO: 22 (= pTEF2.Kl)

  Sequence number 23 (= pTEF3)

  Sequence number 24 (= pADH1)

  SEQ ID NO: 25 (= pGPM1)

  SEQ ID NO: 26 (= pFBA1)

  SEQ ID NO: 27 (= pPDC1)

  SEQ ID NO: 28 (= pPGK1)

  SEQ ID NO: 29 (= pRPLA1)

  Sequence number 30 (= pTEF1)

  SEQ ID NO: 31 (= pTDH3)

  Sequence number 32 (= tTHD2)

  Sequence number 33 (= tCYC1)

  Sequence number 34 (= tTDH3)

  Sequence number 35 (= tADH1)

  SEQ ID NO: 36 (= tTPI1)

  SEQ ID NO: 37 (= tMET25)

  SEQ ID NO: 38 (= tENO2)

  SEQ ID NO: 39 (= tMET3)

  Sequence number 40 (= tPGK1)

  SEQ ID NO: 41 (= pPYK1)

  SEQ ID NO: 42 (= pTPI1)

  Sequence number 43 (= tDIT1)

  Sequence number 44 (= loxP)

  SEQ ID NO: 45 (= nucleic acid HAA-1)

  SEQ ID NO: 46 (= amino acid HAA-1)

  SEQ ID NO: 47 (= nucleic acid LEU2.Kl)

  SEQ ID NO: 48 (= amino acid LEU2.Kl)

  SEQ ID NO: 49 (= nucleic acid His5)

  SEQ ID NO: 50 (= amino acid His5)

  SEQ ID NO: 51 (= nucleic acid Trp1 kl)

  SEQ ID NO: 52 (= amino acid Trp1 Kl)

Claims (25)

Recombinant yeast with reduced pyruvate decarboxylase activity in its genome:
-One or more nucleic acids encoding acetolactate synthase or ALS,
-One or more nucleic acids encoding acetolactate decarboxylase or ALD, and
-Recombinant yeast into which one or more copies of nucleic acid encoding NADH oxidase or NOXE has been inserted. The recombinant yeast of claim 1, comprising one or more DNA constructs selected from the group comprising the following formula:
(I) 5 '-[gene 1] _x1 -3' and 5 '-[gene 2] _x2 -3' and 5 '-[gene 3] _x3 -3',
(II) 5 '-[gene 1] _x1- [gene 2] _x2 -3' and 5 '-[gene 3] _x3 -3',
(III) 5 '-[gene 1] _x1- [gene 2] _x2- [gene 3] _x3 -3', and combinations thereof,
(Where:
-"Gene 1" means a nucleic acid selected from the group comprising ALS, ALD or NOXE;
-"Gene 2" means a nucleic acid selected from the group comprising ALS, ALD or NOXE, but different from gene 1;
-"Gene 3" means a nucleic acid selected from the group comprising ALS, ALD or NOXE, but different from genes 1 and 2;
-"ALS" is a nucleic acid encoding acetolactate synthase;
-"ALD" is a nucleic acid encoding acetolactate decarboxylase;
-"NOXE" is a nucleic acid encoding NADH oxidase;
-Each of "x1", "x2" and "x3", independently of one another, represents an integer in the range of 0 to 50, preferably 0 to 20,
However, the recombinant yeast contains at least one nucleic acid encoding each of ALS, ALD and NOXE).   The recombinant yeast according to claim 2, comprising at least one DNA construct of formula (II), wherein "gene 3" means a nucleic acid encoding NADH oxidase. Recombinant yeast according to claim 2 or 3, comprising at least one, preferably at least two DNA constructs of formula (IIa), identical or different, each formula (IIa) having the following formula:
(IIa) 5 '- [( prom5) y1 - Gene 1-term5] _x5 - [prom1- gene 1-term1] _x1 - [prom2- gene _{2- (term2) z1] x2 -3} ' and 5 '- [( prom3) _y2 -gene 3- (term3) _z2 ] _x3 -3 '
(Where:
-Gene 1, gene 2 and gene 3 are for example as defined in claim 2 or 3, and `` x1 '', `` x2 '' and `` x3 '' are for example as defined in claim 2;
-"X5" represents an integer equal to 0 or 1;
-`` Y1 '', `` y2 '', `` z1 '' and `` z2 '' represent, independently of one another, an integer equal to 0 or 1;
-When the recombinant yeast comprises at least two DNA constructs of formula (IIa), `` x1 '', `` x2 '', `` x3 '', `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '''' May be the same or different;
-"Prom 1" is a regulatory sequence that controls the expression of the sequence encoding gene 1;
-"Prom 2" is a regulatory sequence that controls the expression of the sequence encoding gene 2;
-"Prom 3" is a regulatory sequence that controls the expression of the sequence encoding gene 3;
-`` Prom 5 '' is a control sequence that controls the expression of gene 1, said prom 5 being the same as or different from prom 1;
-"Term 1" is a transcription terminator sequence that terminates the expression of the sequence encoding gene 1;
-"Term 2" is a transcription terminator sequence that terminates the expression of the sequence encoding gene 2;
-"Term 3" is a transcription terminator sequence that terminates the expression of the sequence encoding gene 3;
-"Term 5" is a transcription terminator sequence that terminates the expression of gene 1, and term 5 is the same as or different from term 1). Recombination according to any one of claims 2 to 4, comprising at least one, preferably at least two DNA constructs of formula (IIb), identical or different, each formula (IIb) having the following formula yeast:
(IIb) 5 '- [( prom5) y1 -ALS-term5] x5 - [prom1-ALS-term1] x1 - [prom2-ALD- (term2) z1] x2 -3' and 5 '- [(prom3) _y2 -NOXE- (term3) _z2 ] _x3 -3 '
(Where:
-ALS, ALD and NOXE are for example as defined in any one of claims 2 to 4; `` x1 '', `` x2 '' and `` x3 '' are for example as defined in claim 2; `` X5 '' and `` y1 '', `` y2 '', `` z1 '' and `` z2 '' are for example as defined in claim 4;
-If said recombinant yeast comprises at least two DNA constructs of formula (IIb), `` x1 '', `` x2 '', `` x3 '', `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '' May be the same or different;
-"Prom 1" is a regulatory sequence that controls the expression of the sequence encoding acetolactate synthase;
-"Prom 2" is a regulatory sequence that controls the expression of the sequence encoding acetolactate decarboxylase;
-"Prom 3" is a regulatory sequence that controls the expression of the sequence encoding NADH oxidase;
-"Prom 5" is a control sequence that controls the expression of a sequence encoding acetolactate synthase, said prom 5 being the same as or different from prom 1;
-"Term 1" is a transcription terminator sequence that terminates the expression of the sequence encoding acetolactate synthase;
-"Term 2" is a transcription terminator sequence that terminates the expression of the sequence encoding acetolactate decarboxylase;
-"Term 3" is a transcription terminator sequence that terminates the expression of the sequence encoding NADH oxidase;
“Term 5” is a transcription terminator sequence that terminates the expression of the sequence encoding acetolactate synthase, and the term 5 is the same as or different from the term 1).   Comprises at least two DNA constructs of formula (II), (IIa) or (IIb), provided that all copies of NOXE nucleic acid comprise at least two DNA constructs of formula (II), (IIa) or (IIb) The recombinant yeast according to any one of claims 2 to 5, which is located in one of the above. Recombinant yeast according to any one of claims 2 to 6, comprising at least two, preferably exactly two, DNA constructs of the following formulas (IIc) and (IId):
(IIc) 5 '- [( prom5) y1 -ALS-term5] x5 - [prom1-ALS-term1] x1 - [prom2-ALD- (term2) z1] x2 -3' and 5 '- [(prom3) _y2 -NOXE- (term3) _z2 ] _x6 -3 ';
(IId) 5 '- [( prom5) y1 -ALS-term5] x5 - [prom1-ALS-term1] x1 - [prom2-ALD- (term2) z1] x2 -3' and 5 '- [(prom3) _y2 -NOXE- (term3) _z2 ] _x7 -3 ';
(Where:
-ALS, ALD and NOXE are defined, for example, in any one of claims 2 to 6; `` prom1, '' `` prom2, '' `` prom3, '' `` prom5, '' `` term1, '' `` term2, '' “Term3” and “term5” are, for example, those defined in any one of claims 4 to 5; “x1,” “x2,” and “x3” are, for example, those defined in claim 2. `` X5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '' are, for example, those defined in claim 4;
-`` X1 '' to `` x3 '', `` x5 '', `` y1 '', `` y2 '', `` z1 '' and `` z2 '' are the same or different for each formula (IIc) and (IId); and
-"X6" and "x7" are integers equal to 0 to 50, preferably 0 to 20, preferably 0 to 12, more particularly in the range 2 to 5, preferably 3 to 4, and more preferably 3. Where one of “x6” and “x7” represents 0).   The nucleic acid encoding acetolactate synthase or ALS is a nucleic acid selected from the group comprising Bacillus subtilis, tobacco, Paenibacillus polymixer, and mixtures thereof, and preferably tobacco and Paenibacillus polymixer. Item 8. The recombinant yeast according to any one of Items 1 to 7.   The nucleic acid encoding acetolactate synthase or ALS is a nucleic acid selected from the group consisting of sequences having at least 65%, preferably at least 80% nucleic acid identity with the nucleic acid sequences SEQ ID NOs: 1, 3 and 5. Item 9. The recombinant yeast according to any one of Items 1 to 8.   The nucleic acid encoding acetolactate decarboxylase or ALD is a nucleic acid selected from the group comprising Brevibacillus brevis, Erobacter erogenes, Lactococcus lactis, and mixtures thereof, and preferably Brevibacillus brevis and Erobacter erogenes 10. The recombinant yeast according to any one of claims 1 to 9, wherein   The nucleic acid encoding acetolactate decarboxylase or ALD is a nucleic acid selected from the group consisting of sequences having at least 36%, preferably at least 80% nucleic acid identity with the nucleic acid sequences SEQ ID NOs: 7, 9, and 11. The recombinant yeast according to any one of claims 1 to 10.   The nucleic acid encoding NADH oxidase or NOXE is a nucleic acid selected from the group comprising Streptococcus pneumoniae, Lactococcus lactis, Enterococcus faecalis, Lactobacillus lactis and mixtures thereof, and preferably Streptococcus pneumoniae Item 12. The recombinant yeast according to any one of Items 1 to 11.   The nucleic acid encoding NADH oxidase or NOXE is a nucleic acid selected from the group consisting of sequences having at least 78%, preferably at least 80% nucleic acid identity with the nucleic acid sequences SEQ ID NOs: 21, 23, 25 and 27. The recombinant yeast according to any one of claims 1 to 12.   Each of the nucleic acids encoding acetolactate synthase, acetolactate decarboxylase and NADH oxidase is under the control of the same or different promoter, said promoter comprising at least 80% of the nucleic acid sequences of SEQ ID NOs: 29 to 39, 49 and 50 14. The recombinant yeast according to any one of claims 1 to 13, characterized by the sequence of a nucleic acid selected from the group consisting of sequences having nucleic acid identity.   The first gene in 5'- of the at least one DNA construct is preferably a promoter of a recombinant yeast gene into which the considered DNA construct is inserted when the first gene is a nucleic acid encoding ALS The recombinant yeast according to any one of claims 2, 4, 5 and 7, which is under the control of.   Each of the nucleic acids encoding acetolactate synthase, acetolactate decarboxylase and NADH oxidase is under the control of a transcription terminator, the same or different, said transcription terminator being at least 80% nucleic acid identical to the nucleic acid sequence of SEQ ID NOs: 40-48 16. The recombinant yeast according to any one of claims 1 to 15, characterized by the sequence of a nucleic acid selected from the group consisting of sequences having sex.   The recombinant yeast according to any one of claims 1 to 16, wherein the recombinant yeast belongs to the genus Saccharomyces, preferably a budding yeast species.   18. The recombinant yeast according to any one of claims 1 to 17, wherein pyruvate decarboxylase activity is reduced by disruption of at least one pdc gene.   When the recombinant yeast belongs to the genus Saccharomyces, the pyruvate decarboxylase activity is preferably at least 2 selected from the group consisting of pdc1, pdc5, pdc6 and combinations thereof, more particularly from the group consisting of pdc1 and pdc6. 19. The recombinant yeast according to any one of claims 1 to 18, which is reduced by disruption of one pdc gene.   The pyruvate decarboxylase activity comprises at least one DNA construct selected from the group comprising formulas (I) to (III), and preferably at least one said nucleic acid, preferably comprising only at least one nucleic acid encoding ALS and / or ALD. 20. A recombinant yeast according to claim 18 or 19, which is reduced by insertion of a DNA construct.   The recombinant pyruvate decarboxylase activity is reduced by disruption of the pdc1 gene and the pdc6 gene, and further by attenuation of the pdc5 gene when the recombinant yeast belongs to a budding yeast species. Recombinant yeast.   The DNA construct selected from the group comprising formulas (I) to (III) comprising at least the NOXE gene is inserted into the endogenous URA3 gene of the recombinant yeast, according to any one of claims 2 to 21. Recombinant yeast.   Use of the recombinant yeast according to any one of claims 1 to 22, for example for the production of acetoin and / or derivatives thereof, in particular methyl vinyl ketone. (a) culturing the recombinant yeast as described in any one of claims 1 to 22 in a suitable culture medium; and
(c) A method for producing acetoin, comprising a step of recovering acetoin.   25. A method according to claim 24, wherein the culture medium comprises a carbon source preferably selected from the group comprising glucose and sucrose.